Re: [ccp4bb] Coming July 29: Improved Carbohydrate Data at the PDB -- N-glycans are now separate chains if more than one residue

[Zhijie Li]

Hi Tristan and all,

I totally agree that randomly naming the glycan chains is going to give users 
headaches.  But using more than 2 letters would make the entry incompatible 
with the PDB format, which I wish will remain as a download option for as long 
as possible.  
 How about restricting the length of chain IDs to two characters? Then they can 
be fitted in columns 21 and 22 of PDB files. For example, all N-glycan chains 
associated with chain A can be named 0A...9A, AA,BA, CA. zA. That’s a 
maximum of 62 Nglycans on a chain. If we also use the printable symols that’s 
another 20-30 chains.  
On top of this, if the residues are still each given a residue number shift and 
the “major” letter of the chain ID(one that indicates protein chain) remains on 
column  22, this would mean that the new system might even look the same as the 
old system to the softwares that strictly stick to the PDB format 
specification, as the only change is an extra letter on column 21, which was 
unused.  I think some of the tools are already treating both column 21 and 22 
as chain IDs. Even the pdb deposition system seems to start giving out 2-letter 
IDs when it hits ‘z’.  So this is natural to them and will cause them no 
problem at all.  Does the new carbohydrate standard allow starting a glycan 
chain at residue 5021? 

Zhijie 

> On Dec 4, 2020, at 3:06 AM, Tristan Croll  wrote:
> 
> EXTERNAL EMAIL:
> 
> To go one step further: in large, heavily glycosylated multi-chain complexes 
> the assignment of a random new chain ID to each glycan will lead to headaches 
> for people building visualisations using existing viewers, because it loses 
> the easy name-based association of glycan to parent protein chain. A 
> suggestion: why not take full advantage of the mmCIF capability for 
> multi-character chain IDs, and name them by appending characters to the 
> parent chain ID? Using chain A as an example, perhaps the glycans could 
> become Ag1, Ag2, etc.?
> 
>> On 4 Dec 2020, at 07:48, Luca Jovine  wrote:
>> 
>> CC: pdb-l
>> 
>> Dear Zhijie and Robbie,
>> 
>> I agree with both of you that the new carbohydrate chain assignment 
>> convention that has been recently adopted by PDB introduces confusion, not 
>> just for PDB-REDO but also - and especially - for end users.
>> 
>> Could we kindly ask PDB to improve consistency by either assigning a 
>> separate chain to all covalently attached carbohydrates (regardless of 
>> whether one or more residues have been traced), or reverting to the old 
>> system (where N-/O-glycans inherited the same chain ID of the protein to 
>> which they are attached)? The current hybrid solution hardly seems optimal...
>> 
>> Best regards,
>> 
>> Luca
>> 
>>>> On 3 Dec 2020, at 20:17, Robbie Joosten  wrote:
>>> 
>>> Dear Zhijie,
>>> 
>>> In generally I like the treatment of carbohydrates now as branched 
>>> polymers. I didn't realise there was an exception. It makes sense for 
>>> unlinked carbohydrate ligands, but not for N- or O-glycosylation sites as 
>>> these might change during model building or, in my case, carbohydrate 
>>> rebuilding in PDB-REDO powered by Coot. Thanks for pointing this out.
>>> 
>>> Cheers,
>>> Robbie
>>> 
>>>> -Original Message-
>>>> From: CCP4 bulletin board  On Behalf Of Zhijie Li
>>>> Sent: Thursday, December 3, 2020 19:52
>>>> To: CCP4BB@JISCMAIL.AC.UK
>>>> Subject: Re: [ccp4bb] Coming July 29: Improved Carbohydrate Data at the
>>>> PDB -- N-glycans are now separate chains if more than one residue
>>>> 
>>>> Hi all,
>>>> 
>>>> I was confused when I saw mysterious new glycan chains emerging during
>>>> PDB deposition and spent quite some time trying to find out what was
>>>> wrong with my coordinates.  Then it occurred to me that a lot of recent
>>>> structures also had tens of N-glycan chains.  Finally I realized that this
>>>> phenomenon is a consequence of this PDB policy announced here in July.
>>>> 
>>>> 
>>>> For future depositors who might also get puzzled, let's put it in a short
>>>> sentence:  O- and N-glycans are now separate chains if it they contain more
>>>> than one residue; single residues remain with the protein chain.
>>>> 
>>>> 
>>>> https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.wwpdb.org%2Fdocumentation%2Fcarbohydrate-remediationdata=04%7C01%7Cluca.jovine%40KI.SE%7C1d790a0717ce4217c7a308d897c01b47%7Cbff7eef1cf4b4f32be3da1dda043c05d%7C0%7C1%7C637426199684263065%7CUnknown%7CTW

Re: [ccp4bb] Coming July 29: Improved Carbohydrate Data at the PDB -- N-glycans are now separate chains if more than one residue

[Zhijie Li]

Hi all,

I was confused when I saw mysterious new glycan chains emerging during PDB 
deposition and spent quite some time trying to find out what was wrong with my 
coordinates.  Then it occurred to me that a lot of recent structures also had 
tens of N-glycan chains.  Finally I realized that this phenomenon is a 
consequence of this PDB policy announced here in July.

For future depositors who might also get puzzled, let's put it in a short 
sentence:  O- and N-glycans are now separate chains if it they contain more 
than one residue; single residues remain with the protein chain.

https://www.wwpdb.org/documentation/carbohydrate-remediation
"Oligosaccharide molecules are classified as a new entity type, branched, 
assigned a unique chain ID (_atom_site.auth_asym_id) and a new mmCIF category 
introduced to define the type of branching (_pdbx_entity_branch.type) . "


I found the differential treatment of single-residue glycans and multi-residue 
glycans not only bit lack of aesthetics but also misleading.  When a structure 
contains both NAG-NAG... and single NAG on N-glycosylation sites, it might be 
because of lack of density for building more residues, or because that some of 
the glycosylation sites are now indeed single NAGs (endoH etc.) while some 
others are not cleaved due to accessibility issues.Leaving NAGs on the 
protein chain while assigning NAG-NAG... to a new chain, feels like suggesting 
something about their true oligomeric state.

For example, for cryoEM structures, when one only builds a single NAG at a site 
does not necessarily mean that the protein was treated by endoH. In fact all 
sites are extended to at least tri-Man in most cases. Then why keeping some 
sites associated with the protein chain while others kicked out?

Zhijie




From: CCP4 bulletin board  on behalf of John Berrisford 

Sent: Thursday, July 9, 2020 4:39 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Coming July 29: Improved Carbohydrate Data at the PDB


Dear CCP4BB

PDB data will shortly incorporate a new data representation for carbohydrates 
in PDB entries and reference data that improves the Findability and 
Interoperability of these molecules in macromolecular structures. In order to 
remediate and improve the representation of carbohydrates across the archive, 
the wwPDB has:

  *   standardized Chemical Component Dictionary nomenclature following 
IUPAC-IUBMB recommendations
  *   provided uniform representation for oligosaccharides
  *   adopted Glycoscience-community commonly used linear descriptors using 
community tools
  *   annotated glycosylation sites in PDB structures

Starting July 29, 2020, users will be able to access the improved data via FTP 
or wwPDB partner websites. Detailed information about this project is available 
at the wwPDB 
website; lists of 
impacted entries and chemical components will be published on this page after 
data release.

The wwPDB has created a new ‘branched’ entity representation for 
polysaccharides, describing all the individual monosaccharide components of 
these in the PDB entry. As part of this process, we have standardized atom 
nomenclature of >1,000 monosaccharides in the Chemical Component Dictionary 
(CCD) and applied a branched entity representation to oligosaccharides for 
>8000 PDB entries. To guarantee unambiguous chemical description of 
oligosaccharides in the affected PDB entries, an explicit description of 
covalent linkage information between their monosaccharide units is included. In 
addition, wwPDB validation reports provide consistent representation for these 
oligosaccharides and include 2D representations based on the Symbol 
Nomenclature for Glycans (SNFG).

To support the remediation of carbohydrate representation, software tools 
providing linear descriptors were developed in collaboration with the 
glycoscience community to enable easy translation of PDB data to other 
representations commonly used by glycobiologists. These include Condense IUPAC 
from GMML at University of Georgia, 
WURCS from PDB2Glycan at The Noguchi 
Institute, Japan, and LINUCS 
from pdb-care at Germany.

Furthermore, to ensure continued Findability of 118 common oligosaccharides 
(e.g., sucrose, Lewis Y antigen), we have expanded the Biologically Interesting 
molecule Reference Dictionary (BIRD) that 
contains the covalent linkage information and common synonyms for such 
molecules.

wwPDB has also used this opportunity to improve the organization of chemical 
synonyms in the CCD by introducing a new _pdbx_chem_comp_synonyms data 
category. This will enable more comprehensive capture of alternative names for 
small molecules in the PDB. To minimize disruption to users, the legacy data 
item, 

Re: [ccp4bb] protein expression in human cells

[Zhijie Li]

Hi all,

If anybody is interested in non-viral stable expression, we have a 
piggybac-based, doxycycline-inducible system. It is the reference 40 in the 
lentiviral paper that Tomas directed to.  We’d be happy to distribute the 
plasmids.

Zhijie


> On Jan 25, 2020, at 3:26 AM, Tomas Malinauskas  
> wrote:
> 
> Hi Gloria,
> 
> two key papers describing expression (transient and lentivirus-based)
> of proteins in HEK293 cells we use to make milligrams of proteins for
> crystallization and cryo-EM:
> 
> Acta Crystallogr D Biol Crystallogr. 2006 Oct;62(Pt 10):1243-50. Epub
> 2006 Sep 19.
> A time- and cost-efficient system for high-level protein production in
> mammalian cells.
> Aricescu AR, Lu W, Jones EY.
> PMID: 17001101
> 
> Nat Protoc. 2018 Dec;13(12):2991-3017. doi: 10.1038/s41596-018-0075-9.
> Lentiviral transduction of mammalian cells for fast, scalable and
> high-level production of soluble and membrane proteins.
> Elegheert J, Behiels E, Bishop B, Scott S, Woolley RE, Griffiths SC,
> Byrne EFX, Chang VT, Stuart DI, Jones EY, Siebold C, Aricescu AR.
> PMID: 30455477
> 
> Hope that helps,
> Tomas
> 
> 
> Dr. Tomas Malinauskas
> University of Oxford
> Wellcome Centre for Human Genetics
> Division of Structural Biology
> Roosevelt Drive
> Oxford OX3 7BN
> United Kingdom
> to...@strubi.ox.ac.uk
> tomas.malinaus...@gmail.com
> 
>> On Fri, Jan 24, 2020 at 11:50 PM Gloria Borgstahl  
>> wrote:
>> 
>> Hello CCP4-ers,
>> I was wondering what people have found to be the best human cell line 
>> expression system for making a large quantity of purified recombinant 
>> protein.
>> Any information and protocols would be greatly appreciated.
>> Happy 2020, Gloria
>> 
>> 
>> 
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> 
> 
> 
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Re: [ccp4bb] Generating symmetry mates using python

[Zhijie Li]

Hi Orly,

REMARK 290 should be the easiest way for generating symmetry mates. Other 
routes are just going to give you the same results. As Jonathan already pointed 
out, the symm ops do not garantee that the symm copies are close to each other. 
 The most simple-minded solution to this problem would be simply generating 
3x3x3 unit cells so that the unit cell in center will be complete. An upgrade 
to this is to compute the center of mass of the symmetry copies in each of the 
3x3c3 cells and find which one is closest to the orignal 1555 copy.  Just for 
fun, I wrote a little python script that does this (attached). In this script 
for unit cell translation and calculating center-center distances, I converted 
the Cartesian coordinates to fractional coords first. Then after the 
translation,I used the inverse of the SCALE1 matrix to get the shifted 
Cartesian coords. This way I don't need to read wikipedia on geometry . But as 
noted in the script the distances should better be calculated in Cartesian.

Zhijie


From: CCP4 bulletin board  on behalf of orly avraham 

Sent: Friday, January 10, 2020 3:30 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Generating symmetry mates using python

Hi all,

I am a crystallographer currently employing computational methods as well as 
experimental crystallography.
I am trying to generate symmetry mates in python (working with pandas 
dataframes), in order to analyze inter-sub-unit interactions. To do so I am 
trying to use the info in "REMARK 290 CRYSTALLOGRAPHIC SYMMETRY" and manually 
(using numpy) perform a matrix multiplication with the relevant translation 
(xyz*rotation + translation).
For some reason this doesn't work consistently and I feel I need to use the 
info in CRYST1 to obtain the unit cell and multiplication matrix. Here I ran 
into trouble with extracting the correct symmetry operations based on each 
space group. I found spglib but it doesn't quite solve the problem.
I also tried opening PyMol through the command and generating symmetry mates 
this way. It worked on a few files but failed quite quickly (segmentation 
fault) and was also very slow.
Can anyone suggest a useful solution, preferably clear to use and/or well 
documented? Or even have a python script/code they can share for this?

Best regards,
Orly

--

Orly Avraham, Ph.D.
Postdoctoral fellow
The lab of Prof. Oded Livnah
and the lab of Prof. Ora Schueler-Furman
The Hebrew University of Jerusalem
Israel




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py_PDB.py
Description: py_PDB.py

Re: [ccp4bb] microscope camera

[Zhijie Li]

There are CCD modules (often come with lenses, which can be taken off) sold on 
Amazon, eBay or aliexpress (or even better, Taobao) costing USD 20-40. These 
modules should be capable of taking full HD movies (MPEG or YUV2 compressed) at 
30fps these days. Some may allow 120fps@640x480 or promise higher sensitivities.

A better alternative is old DSLR bodies, which have larger, better sensors. 
There should be plenty of them, cheaply, on 2nd-hand market. The aforementioned 
CCD modules all use the smallest, lowest-end (but OK for what we do) sensors, 
in constrast.


Then with a 3D printer and some black PLA filament it is fairly easy to make a 
custom mounting tube that directly puts the CCD or the camera sensor on the 
image plane of the microscope. Metal adaptors are also available for DSLR 
bodies.

To control the CCD modules through USB, one can use amcap or VLC player. For 
DSLR bodies, digiCamControl. If one has a Raspberrypi, on-line streaming can 
also be setup using mjpg-streamer (or simply use the streaming function of 
octoprint).

Zhijie


From: CCP4 bulletin board  on behalf of Dean Derbyshire 

Sent: Wednesday, November 27, 2019 11:35
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] microscope camera

Forgive the off topic subject:

Has anyone got any experience with moticam microscope cameras?
We are looking into cheap cameras for record keeping etc and this supplier 
looks good but…

Cheers in advance

Sent from Mail for Windows 10




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Re: [ccp4bb] More LCF archaeology (unit cell parameters this time).

[Zhijie Li]

Hi Jonathan,

We discussed about VAX floats last November:

https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1811=CCP4BB=D=63307

https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1811=CCP4BB=D=65015

https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1811=CCP4BB=D=65531

In short, the factor of 4 comes from: a) a bias of 128 instead of 127, b) a 
different convention in the scientific notation.

Would you be so kind as to send me a specimen of the VAX LCF file off-list? I 
am also quite interested in archaeology.

Zhijie




From: CCP4 bulletin board  on behalf of Jonathan Cooper 
<0c2488af9525-dmarc-requ...@jiscmail.ac.uk>
Sent: Saturday, July 13, 2019 4:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] More LCF archaeology (unit cell parameters this time).

I am trying to use a hex editor to read the unit cell parameters from the 
headers of 80's VAX LCF files and I can definitely find them in front of the 
column labels as six regularly spaced 32-bit floats. However, they seem to be 
multiplied by a factor of 4 and the final corrected values of a, b, c, alpha, 
beta and gamma are a bit more approximate than I would expect. I can't work out 
what's going on from the fortran yet so any clues would be much appreciated.



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Re: [ccp4bb] beryllium chloride

[Zhijie Li]

Sometime ago when I was watching a Youtube video on magnetron I learnt that at 
least at at certain point of time the antenna port of the magnetron was sealed 
using beryllium oxide ceramic(probably for its high thermal conductivity). The 
video maker warned that this ceramic was extremely dangerous. Further wikipedia 
and internet reading confirmed that fine beryllium oxide powder does cause 
something called beryllium disease and cancer (the latter in a way similar to 
that of asbestos? I guessed). However the sintered ceramic form is probably as 
safe as emerald unless you have to grind it and breath the dust every day. (I 
think most jewellers would always use some grinding liquid when honing their 
stones.)
The wikipedia page on beryllium oxide has some really interesting facts. Also 
if one is desperate for some beryllium salt it hints dissolving a magnetron 
antenna sealing ring in hot concentrated solution of H2SO4 and (NH4)2SO4.

My 2 cents.

Zhijie



On Apr 2, 2019, at 8:39 AM, Ian Tickle 
mailto:ianj...@gmail.com>> wrote:


Yes both soluble beryllium salts and powdered beryllium metal even applied to 
the skin are known to cause sensitization and is a route into the bloodstream 
where it is highly carcinogenic (I am not speaking from experience!).

Yet strangely the one source of beryllium that many people (at least the more 
well-off among us) commonly come into contact with, namely the gemstone emerald 
Be3Al2(SiO3)6 obviously has no known toxic effects whatosever!  Apparently even 
gemstone grinders show no ill effects!  I guess it's the free Be2+ ion that's 
so toxic.

Cheers

-- Ian



On Tue, 2 Apr 2019 at 13:07, Aaron Finke 
mailto:af...@cornell.edu>> wrote:
1. Yes, I meant the tetrahydrate, [Be(H2O)4]2+ 2Cl-

2. Bob, I studied that page, and couldn’t get past  BeCl2 is known to have a 
“sweetish taste.” I’m very glad chemists no longer characterize chemicals by 
their taste anymore...

--
Aaron Finke
Staff Scientist, MacCHESS
Cornell University
e-mail: af...@cornell.edu

On Apr 1, 2019, at 22:37, Sweet, Robert 
<27e0eb9d20ec-dmarc-requ...@jiscmail.ac.uk>
 wrote:


With all respect, this conversation make my skin crawl a little. I've been 
taught that beryllium salts are EXTREMELY toxic.  Please study this: 
https://pubchem.ncbi.nlm.nih.gov/compound/beryllium_chloride


Hopefully,


Bob



  Robert M. Sweet   E-Dress:  
sw...@bnl.gov
  Deputy Director, LSBR: The Life Science and
   Biomedical Technology Research Center at NSLS-II
  Photon Sciences and Biology Dept
  Brookhaven Nat'l Lab.
  Upton, NY  11973 U.S.A.
  Phones:631 344 3401  (Office)
 631 338 7302  (Mobile)



From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Diana Tomchick 
mailto:diana.tomch...@utsouthwestern.edu>>
Sent: Monday, April 1, 2019 7:03 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] beryllium chloride

No, that should read



Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
UT Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

On Apr 1, 2019, at 5:54 PM, Keller, Jacob 
mailto:kell...@janelia.hhmi.org>> wrote:

Is that 4+ an April fools’ joke? Pretty crazy if not…can’t think of another ion 
with such a charge, well except things like DNA and proteins, but not single 
atoms.

JPK

+
Jacob Pearson Keller
Research Scientist / Looger Lab
HHMI Janelia Research Campus
19700 Helix Dr, Ashburn, VA 20147
Desk: (571)209-4000 x3159
Cell: (301)592-7004
+

The content of this email is confidential and intended for the recipient 
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received this message by mistake, please reply to this message and follow with 
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From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Aaron Finke
Sent: Monday, April 1, 2019 6:45 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] beryllium chloride

American Elements sells BeCl2 but you’d have to check with them on what scale 
they sell it at. They tend to do custom manufacturing.

https://www.americanelements.com/beryllium-chloride-7787-47-5

BeCl2 dissociates in aqueous solution to form Be(H2O)4+ 2Cl-.


Aaron

Re: [ccp4bb] map rotation

[Zhijie Li]

Hi Jan,


As Paul pointed out, you can use COOT to accomplish what you want. Particularly 
you can take a look at the following functions in section 6 of the COOT manual 
(these are in scheme):



(set-map-mask-atom-radius radius)


(mask-map-by-molecule imol-map imol-model invert-mask?)


(transform-map imol rotation-matrix trans point radius)

(transform-map-using-lsq-matrix imol-ref ref-chain ref-resno-start 
ref-resno-end imol-mov mov-chain mov-resno-start mov-resno-end imol-map 
about-pt radius)




Please take a look at the attached python example.  It can be run with COOT 
graphical interface by:


coot scr.py


You can run it without COOT graphical interface by un-commenting the exit() 
line at the end, then:


coot --no-graphics -s scr.py

If you see any part of the resulting transformed map being cut in the protein 
region, play with the rotation center (supply your coordinates [x,y,z] to 
replace the rotation_center() function.  In this script, the current rotation 
center is set upon reading the last pdb.).


BTW, if you see jagged map after rotation, that's probably because the map 
sampling frequency before the rotation is too low. When you rotate a map, there 
is always a resolution loss, because of the rastering nature of the maps 
(imagine rotating a mesh then resample it to become another mesh of the same 
spacing, how can you keep all the information?). So you had better use a very 
high sampling frequency when you are preparing a map that is going to be 
rotated. Since we often start from a MTZ file, you may even start by computing 
the starting map by specifying the sampling frequency to be more than 5x of the 
highest resolution to be very safe. On the other hand, if you use COOT i guess 
an interpolation is going to be done internally before the rotation (however 
interpolation is, maybe to some degree, not as good as computing a very 
over-sampled map from MTZ, I think.).


Zhijie



From: CCP4 bulletin board  on behalf of Jan Abendroth 

Sent: Tuesday, March 26, 2019 2:05 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] map rotation

Thanks, Eleanor!
what i really want to do with this script is to compare ligand binding sites 
from many structures in several space groups. So, I want to move coordinates 
and density onto one reference molecule. Since we ran into issues, I used this 
dimer structure as a simple test case.

The script you outlined, only recreates the same density w/o any rotation.

mapmask \

mapin AB_full-cell.ccp4 \

xyzin B.pdb \

mskout B.msk \

<< eof

border 5

eof


maprot \

mapin AB_full-cell.ccp4 \

mskin B.msk \

mapout B_rot.map \

<< eof

MODE to

AVER

rota euler 152.440   110.24328.112

TRANS  -42.212 5.510   -57.243

end

eof


Btw, I had to remove the rota euler 0 0 0 / trans 0 0 0 lines. Maprot 
complained about too many operators.

The odd thing -to me- is that when I shift a map around molecule B or the dimer 
(mapmask with border 5) with a small amount ( euler 1 0 0  or trans 0.5 0.5 
0.5) the map does not look jagged at the edges of the molecule, while it does 
when I rotate the full amount to match B on A:

maprot  \

wrkin  AB-5.map \

mapout AB_rot.map \

 << eof

CELL xtal 61.0100   142.360068.280090.97.198090.

GRID xtal 100 228 112

MODE to

AVER

rota euler  1 0 0

TRANS   0 0 0

end

eof

Cheers,
Jan

On Mon, Mar 25, 2019 at 3:49 AM Eleanor Dodson 
mailto:eleanor.dod...@york.ac.uk>> wrote:
Hmm - I find maprot extremely confusing, but remember a wrkmap does not use any 
symmetry so maybe that is why some is lost.

I would have done this, but I havent tested it. And the documentation is 
SERIOUSLY confusing!!

Do I understand you want to ADD the density for mol B to that of Mol A


mapmask mapin whole-cell.map
xyzin A.pdb
mskout A-mol.msk

Then
maprot
mapin whole-cell.map
mskin A-mol.msk ! only density withing this mask is 
interesting
mapout A-mol.map

MODE to

AVER

rota euler 0 0 0! to pick up mol A density

trans 0 0 0


rota euler 152.440   110.24328.112   ! to rotate map by B to A rotation

TRANS  -42.212 5.510   -57.243

end




On Mon, 25 Mar 2019 at 04:58, Jan Abendroth 
mailto:jan.abendr...@gmail.com>> wrote:
Hi all,
thanks for the feedback. Suggestions like coot or pymol won't work for us well, 
since we will have to do this with dozens of structures/maps.So, I'd rather 
have this scripted.

Still running into some issues that I think relate to maprot.
My understanding is that I first have to create a map covering molecule B that 
I want to map on A. Checking the extend of the map in chimera confirms that 
this worked:


mapmask \

mapin 2mol_2mFo-DFc.map \

xyzin 2mol_B.pdb \

mapout 2mol_2mFo-DFc_B.map \

<< eof

border 5

eof


Next, I need to rotate/translate the map in maprot. Since in maprot, mapin 
requires a map that covers the unit cell, I use wrkin and 'mode to' as below. 
In this script, 

Re: [ccp4bb] map rotation

[Zhijie Li]

Hi Jan,


Sorry I didn't read your script earlier. If you change your mapmask command to 
output a map instead of a mask it may work for you:


mapmask \

mapin 2mol_2mFo-DFc.map \

xyzin 2mol_B.pdb \

MAPOUT B.map \

<< eof

border 2

eof


then you should get a map that covers the whole molecule B, and whatever 
happens to share the parallelepiped volume with it. This is essentially what 
the FFT method I mentioned in previous email does: modify the origin and the 
extent of the output map to cover the PDB, not really masking the map based on 
the atoms.


If you want to further mask the map to cover atoms of B only, then you can use 
UCSF chimera -- volume viewer -- zone to selected atoms --save as mrc density 
map (same as ccp4 map, just different extension and no crystallographic SG). 
Some shifting may be needed, but it should be trivial.


I also did a little test with the mskout option of mapmask. The generated mask 
file does look strange, or, not what a naive user would expect maybe?


Zhijie



From: CCP4 bulletin board  on behalf of Jan Abendroth 

Sent: Thursday, March 21, 2019 12:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] map rotation

Hi all,
this should be easy, scripting the rotation of a map.
Purpose for this is: Superimpose several structures of the same protein that 
crystallized in different space groups, and then drag the maps along.
As a simple test, I took a dimeric protein and try to superimpose molecule B 
along with the map on molecule A.

The execution should be straightforward:
a) take a map that covers the unit cell (fft),
b) generate a mask around molecule B (mapmask),
c) apply rotation/translation that I obtain from superimposing molecule B on 
molecule A.

The issue is that the obtained map covers both molecule A and B (not a big 
deal), more importantly, it cuts of certain areas on both molecules. Molecule A 
and B have low RMSDs (0.5Å).

I must be missing something fairly obvious, have not been able to see what. 
Feedback would be much appreciated. Scripts are below.

Thanks!
Jan


mapmask \

mapin 2mol_2mFo-DFc.map \

xyzin 2mol_B.pdb \

mskout 2mol_2mFo-DFc_2mol_B.msk \

<< eof

border 2

eof


maprot  \

mapin  2mol_2mFo-DFc.map \

mskin 2mol_2mFo-DFc_2mol_B.msk \

wrkout 2mol_2mFo-DFc_rot.map \

 << eof

MODE from

AVER

ROTA euler  152.440   110.24328.112

TRANS -42.212 5.510   -57.243

eof

--
Jan Abendroth
Seattle / Bainbridge Island, WA, USA
home: Jan.Abendroth_at_gmail.com




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Re: [ccp4bb] map rotation

[Zhijie Li]

Hi Jan,
I guess you might be seeing a flat cut surface on the maps? It might be that 
the map’s extent is not covering the protein molecule, which is usually the 
case. When you rotate crystallographic maps it is usually no longer possible to 
take adjacent unit cells to continue the density on the boundaries as the 
crystallographic periodicity is lost.

You may try computing maps that cover “all atoms in PDB “ using the map tool 
(FFT) from the ccp4 interface. Maps generated this way cover your protein 
portion supplied in the PDB file. Alternatively you can measure the extremes of 
your A and B monomers and change the extents of the maps by hand.

BTW, UCSF chimera does not automatically extend maps as COOT does. So you can 
easily see what is the extent of your current map using chimera.

Zhijie

On Mar 21, 2019, at 12:27 AM, Jan Abendroth 
mailto:jan.abendr...@gmail.com>> wrote:

Hi all,
this should be easy, scripting the rotation of a map.
Purpose for this is: Superimpose several structures of the same protein that 
crystallized in different space groups, and then drag the maps along.
As a simple test, I took a dimeric protein and try to superimpose molecule B 
along with the map on molecule A.

The execution should be straightforward:
a) take a map that covers the unit cell (fft),
b) generate a mask around molecule B (mapmask),
c) apply rotation/translation that I obtain from superimposing molecule B on 
molecule A.

The issue is that the obtained map covers both molecule A and B (not a big 
deal), more importantly, it cuts of certain areas on both molecules. Molecule A 
and B have low RMSDs (0.5Å).

I must be missing something fairly obvious, have not been able to see what. 
Feedback would be much appreciated. Scripts are below.

Thanks!
Jan


mapmask \

mapin 2mol_2mFo-DFc.map \

xyzin 2mol_B.pdb \

mskout 2mol_2mFo-DFc_2mol_B.msk \

<< eof

border 2

eof


maprot  \

mapin  2mol_2mFo-DFc.map \

mskin 2mol_2mFo-DFc_2mol_B.msk \

wrkout 2mol_2mFo-DFc_rot.map \

 << eof

MODE from

AVER

ROTA euler  152.440   110.24328.112

TRANS -42.212 5.510   -57.243

eof

--
Jan Abendroth
Seattle / Bainbridge Island, WA, USA
home: Jan.Abendroth_at_gmail.com




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Re: [ccp4bb] TEV Protease in low reducing agent?

[Zhijie Li]

Hi Nicola,

0.1-1mM Cysteine, GSH or BME will work fine.  You can also try using very fresh 
TEV without reducing reagent (or store it with BME and remove the reducing 
reagent by some column just before use). Depending on how concentrated the 
stock is, diluting it 3-5x could also sufficiently reduce the redox potential 
of the solution. Another thing to try is to add some GS-SG to consume the DTT 
if it is already there.
TEV is not a very stable protease due to both oxidation and aggregation. So 
yes, there is a strong reason to make it in the lab. We can usually make 
20-40mg crude TEV from a liter of LB culture.

Zhijie

> On Feb 13, 2019, at 1:47 PM, Nicola Evans 
> <251ca3b4615e-dmarc-requ...@jiscmail.ac.uk> wrote:
> 
> I am interested in purifying proteins with 1 or 2 disulphide bonds, and have 
> been using enterokinase to cleave off N-terminal tags but we have had issues 
> with poor cleavage and want to try TEV cleavage instead. However TEV protease 
> is usually kept in a high amount of DTT and I am concerned about reducing the 
> disulphide bonds in my purified proteins. I have used TEV protease before 
> with 0.25mM TCEP and it worked well. Is there a way to use TEV with very 
> little or no reducing agent? Perhaps by optimising conditions such as adding 
> glycerol? We were thinking of buying in commercial TEV protease so any advice 
> on that is also welcomed (is there any merit, except cost, to making it 
> ourselves?)
> 
> 
> 
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Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] Is there any alternative to siliconized glass coverslips for crystallization?

[Zhijie Li]

Hi,

I believe that one can put a 50-100uL drop of  fresh SigmaCote (in a tube cap) 
with the glass pieces (surface well exposed), sealed in a dedicated (because 
the container will be coated too) container (air-tight lunch boxes).  After a 
while the SigmaCote vapor should react with the glass and bring polysiloxane 
groups to the glass surface. I have done this with microscope slips. To make a 
few hundred .22 mm cover slips I think the major challenge is to make a frame 
for supporting the separated (~1mm apart to allow free diffusion?) cover slips 
(3D print?). Dry paper may work too. If some of the cover slips tend to stick 
together, a little vacuuming can help.

SigmaCote is a chlorinated polysiloxane. The Cl-Si group allows it to react 
with the hydroxyls on glass surface. Just be aware that the same group reacts 
even more readily with water in air, making the reagent less reactive with 
glass overtime (but I have used a VERY old bottle and it worked).

References:

https://en.wikipedia.org/wiki/Chlorosilane

https://pubs.acs.org/doi/pdfplus/10.1021/j100727a046

https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Product_Information_Sheet/1/sl2pis.pdf

Zhijie


On 31/01/2019 4:16 a.m., 
herman.schreu...@sanofi.com wrote:
A long time ago, before siliconized coverslips became commercially available, 
we used to siliconize coverslips ourselves. It is not really that much work and 
unsiliconized cover slips should be very cheap. If you wish, I could try to 
find back the protocol.
Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Rajnandani Kashyap
Gesendet: Donnerstag, 31. Januar 2019 09:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [EXTERNAL] [ccp4bb] Is there any alternative to siliconized glass 
coverslips for crystallization?

Dear All
I am a PhD student who requires lots of coverslips (!!) for setting up hanging 
drop crystallization. The company sells it for a huge amount. Also there is a 
wide monetary difference between a normal siliconized coverslip and a 22mm 
siliconized circle coverslips. We tried to search for an alternative companies 
but couldn't get any one who sells coverslips with the same dimensions 
(0.19-0.22mm glass thickness and 22 mm glass diameter). Is there any 
alternative company (distribution in India) from where we can buy them for a 
reasonable price?
Thanks in advance for sparing your valuable time and efforts.

Regards
Rajnandani Kashyap
India



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Re: [ccp4bb] Off topic question

[Zhijie Li]

PCA?

On Jan 3, 2019, at 3:41 PM, Reza Khayat 
mailto:rkha...@ccny.cuny.edu>> wrote:


​Hi,


Happy new year to all!  A bit of an off topic question.  Does anyone know of a 
method/program to extract the most distinct "n" (n>2) sequences from a sequence 
alignment?  Thanks.


Best wishes,
Reza


Reza Khayat, PhD
Assistant Professor
City College of New York
Department of Chemistry
New York, NY 10031



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Re: [ccp4bb] Non-specific disulfides in a secreted protein

[Zhijie Li]

Yes, we have also seen some indication that overdriving the expression can 
cause problems. In our cases this may range from bad aggregations or loss of 
expression. Since we use induced stable cells we simply reduced the doxycyline 
levels.
Zhijie

> On Dec 14, 2018, at 1:13 PM, "r...@mrc-lmb.cam.ac.uk" 
>  wrote:
> 
> Hi Tomas,
> 
> I have seen something similar in the past, see PMID: 26998761. The problem
> could be alleviated by tuning down expression levels, through plasmid
> dilution. I guess that cellular QC mechanisms are overwhelmed if you push too
> hard for overexpression. I'd test a range of 1:10 to 1:1000, or higher,
> dilutions in empty (pLEXm or any irrelevant plasmid) to keep total DNA amounts
> constant. Is there no hint of monomer whatsoever in your prep?
> 
> Best wishes,
> 
> Radu
> 
> -- 
> Radu Aricescu
> MRC Laboratory of Molecular Biology
> Francis Crick Avenue
> Cambridge Biomedical Campus
> Cambridge CB2 0QH, U.K.
> tel: +44-(0)1223-267049
> fax: +44-(0)1223-268305
> www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu
> 
>> Dear All,
>> 
>> we are purifying a small secreted protein from conditioned media and
>> have a rather unusual problem.
>> 
>> It is a small ectodomain (~11 kDa, pI ~6, His-tagged) of the type 1
>> transmembrane receptor, crystal structures are known (of the protein
>> that was produced in E.coli and refolded; we are secreting the same
>> protein using mammalian cells) so we can design reasonable constructs.
>> The protein is expressed and secreted by transiently transfected
>> HEK293T cells that work very well for other ectodomains and
>> extracellular proteins in our hands (PMID 17001101). The target
>> protein has 10 cysteines that form 5 disulfides in the crystal
>> structure (of E.coli-expressed and refolded protein), there should be
>> no free cysteines and no non-specific disulfides. Unfortunately, once
>> the protein is secreted, it forms non-specific dimers and higher-order
>> oligomers in the media (standard DMEM/2% FBS) before purification
>> (confirmed by Western blotting under non-reducing conditions). Using
>> 0.5 mM DTT during SEC gives a nice monomeric peak (however, the
>> protein suffers as suggested by weaker interactions with its binding
>> partners). We don't understand how a secreted protein (which passes
>> trafficking quality control in the cell) with a known disulfide
>> pattern forms non-specific disulfide linked oligomers in the
>> extracellular media. We tried expressing it at 37 C and 30 C, and have
>> sequenced our constructs (plasmids) multiple times.
>> 
>> If anyone has seen this kind of problem and successfully solved it
>> (purified homogeneous crystallisation quality protein), please let us
>> know if possible. I thank you for your help.
>> 
>> Best wishes,
>> Tomas
>> 
>> 
>> Dr. Tomas Malinauskas
>> University of Oxford
>> Wellcome Centre for Human Genetics
>> Division of Structural Biology
>> Roosevelt Drive
>> Oxford OX3 7BN
>> United Kingdom
>> to...@strubi.ox.ac.uk
>> tomas.malinaus...@gmail.com
>> 
>> 
>> 
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>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>> 
> 
> 
> 
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Re: [ccp4bb] Non-specific disulfides in a secreted protein

[Zhijie Li]

Hi Tomas,
Some thoughts:
a) I guess the thermodynamic drive for all part of this small ectodomain to 
fold into a single lowest energy conformation is not very strong. The cells 
can’t know that the little artificial domain is supposed to be a monomer when 
the oligomers are also sufficiently hydrophilic.  We also occasionally see 
aggregates coming out of human cell lines. Sometimes barely anything good.

(One wild thought: the disulfide shuffling system in eukaryotic cells have 
something to do with the N-glycans. Does the natural form of this protein have 
N-glycans, while the ecotodomain is somehow deprived of that?)

b) Is there absolutely no monomer on the Western? If this is the case, since 
the bacterial preps can be refolded, can you try incubating the protein with 
Cysteine or GSH or even add PDI or DsbC to try to increase the population of 
properly folded species?

c) If you have some monomers or can manage to generate some by disulfide 
shuffling: for a small domain with such high disulfide content a further 
reverse-phase C18 HPLC step may give you the properly folded species without 
irreversibly denaturing them. Or maybe a Superdex75 gel filtration or a silica 
SEC on HPLC, without reducing reagents, see below.

d) Have you ever run the gel filtration without DTT? When putting the DTT, 
because of its strong disulfide cleaving power, you might be sending the 
misfoled junk to the monomer peak. They look like monomers when DTT is present 
but they are misfolded still. If you have a small fraction of monomers that had 
passed the cell’s QC, they are more likely to be correctly folded therefore 
active. Then the right thing to do I think is to try to separate the monomers 
from the oligomers, rather than forcing everything into “monomers”.

e) Try a larger tag? Add more natural sequence back to the domain? Try 
periplasmic expression in E coli? The NEB pMal-p vector is my favourite for 
disulfide forming proteins, when not using mammalian cells.

Zhijie

> On Dec 14, 2018, at 11:57 AM, Tomas Malinauskas  
> wrote:
> 
> Dear All,
> 
> we are purifying a small secreted protein from conditioned media and
> have a rather unusual problem.
> 
> It is a small ectodomain (~11 kDa, pI ~6, His-tagged) of the type 1
> transmembrane receptor, crystal structures are known (of the protein
> that was produced in E.coli and refolded; we are secreting the same
> protein using mammalian cells) so we can design reasonable constructs.
> The protein is expressed and secreted by transiently transfected
> HEK293T cells that work very well for other ectodomains and
> extracellular proteins in our hands (PMID 17001101). The target
> protein has 10 cysteines that form 5 disulfides in the crystal
> structure (of E.coli-expressed and refolded protein), there should be
> no free cysteines and no non-specific disulfides. Unfortunately, once
> the protein is secreted, it forms non-specific dimers and higher-order
> oligomers in the media (standard DMEM/2% FBS) before purification
> (confirmed by Western blotting under non-reducing conditions). Using
> 0.5 mM DTT during SEC gives a nice monomeric peak (however, the
> protein suffers as suggested by weaker interactions with its binding
> partners). We don't understand how a secreted protein (which passes
> trafficking quality control in the cell) with a known disulfide
> pattern forms non-specific disulfide linked oligomers in the
> extracellular media. We tried expressing it at 37 C and 30 C, and have
> sequenced our constructs (plasmids) multiple times.
> 
> If anyone has seen this kind of problem and successfully solved it
> (purified homogeneous crystallisation quality protein), please let us
> know if possible. I thank you for your help.
> 
> Best wishes,
> Tomas
> 
> 
> Dr. Tomas Malinauskas
> University of Oxford
> Wellcome Centre for Human Genetics
> Division of Structural Biology
> Roosevelt Drive
> Oxford OX3 7BN
> United Kingdom
> to...@strubi.ox.ac.uk
> tomas.malinaus...@gmail.com
> 
> 
> 
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Re: [ccp4bb] VERY old mtz file..

[Zhijie Li]

Hi Nick,


I think I've found the machine stamps in the ccp4 lib (Ian Tickle directed me 
to the C programs folder yesterday. Thanks Ian):

ccp4_ssydep.h

#define DFNTI_MBO   1   /**< Motorola byte order 2's compl */
#define DFNTI_IBO   4   /**< Intel byte order 2's compl */

#define DFNTF_BEIEEE1   /**< big endian IEEE (canonical) */
#define DFNTF_VAX   2   /**< Vax format */
#define DFNTF_CONVEXNATIVE 5/**< Convex native floats */
#define DFNTF_LEIEEE4   /**< little-endian IEEE format */


Checking the numbers are quite feasible for MTZ files (not maps because we can 
put anything into MRC/ccp4 map). Yes, the idea is to try all possibilities 
until we can recover miller indices that look normal.

Zhijie


From: Nicholas Devenish 
Sent: Wednesday, November 14, 2018 9:29 AM
To: Zhijie Li
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] VERY old mtz file..

Hi Zhijie,

Thanks for the answer. I'd read http://www.ccp4.ac.uk/html/mtzformat.html "The 
first 4 half-bytes represent the real, complex, integer and character formats, 
and the last two bytes are currently unused" - and assumed that a) formats 
meant size, given that it was (4,4,4,1) in files I'd seen, though perhaps 
parsers don't really seem to use this. and that b) while this doesn't specify 
endian-ness, one could infer it from whether the two unused zero-bytes came in 
the little-or-big end of the integer. Otherwise there really isn't an encoded 
way to tell if the file was written little or big other than guessing and 
checking if the numbers are sensible?
CCP4 Program Suite: mtz format<http://www.ccp4.ac.uk/html/mtzformat.html>
www.ccp4.ac.uk
Column types. All columns in an MTZ file are assigned a type, taken from the 
following list. The LABIN line of a particular job connects columns in an input 
MTZ file with the columns expected by the program.




Perhaps this is a (small) flaw in the spec, though nowadays almost everyone 
seems to have moved to little-endian.

Nick

On Wed, Nov 14, 2018 at 2:13 PM Zhijie Li 
mailto:zhijie...@utoronto.ca>> wrote:
>
> Hi Nick,
>
>
> I guessed the machine stamp from MRC/CCP4 format description - half byte 01 
> means BE, half byte 04 means LE. Are these only applicable to intel machines? 
> How are other machine architectures indicated? I do not know. We probably can 
> find the authoritative answer from the CCP4 library, just need a little bit 
> more time...
>
>
> The machine stamp itself should not be affected by the machine's 
> architecture, because it needs to be read before the program knows what the 
> architecture is. Therefore it should be a string instead of a number. In the 
> MRC/CCP4 map specification, it says that only the first two bytes are used.  
> I had seen some home-brew programs taking shortcuts by using the int value of 
> the machine stamp word, and I thought that was smart. Now I realise that this 
> practice has the risk of failing on non-intel machines. So, if it is meant to 
> be half-bytes, interpret it as half bytes!
>
>
> Zhijie
>
>
>
> ________
> From: Nicholas Devenish mailto:ndeven...@gmail.com>>
> Sent: Wednesday, November 14, 2018 8:54 AM
> To: Zhijie Li
> Cc: CCP4BB@jiscmail.ac.uk<mailto:CCP4BB@jiscmail.ac.uk>
> Subject: Re: [ccp4bb] VERY old mtz file..
>
> Hi Zhijie,
>
> Looks like we both had the same thoughts!
>
> On Wed, Nov 14, 2018 at 1:19 PM Zhijie Li 
> mailto:zhijie...@utoronto.ca>> wrote:
>
> The semiBE.mtz has the big endian stamp (0x11 11 00 00 at bytes 9-12 ) 
> put in the header of the original file; everything else is untouched
>
>
> Is this correct? I thought machine stamp was supposed to be the 4-bit sizes 
> of int, real, complex and char e.g. all the little endian MTZ's I've seen 
> have 0x44 41 00 00, which would make the big-endian version 0x00 00 41 44. 
> Then again, mtzdmp complained regardless, and perhaps I've just 
> misinterpreted the documentation (I'd love to know if this was the case!)
>
> Thanks,
>
> Nick
>



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Re: [ccp4bb] VERY old mtz file..

[Zhijie Li]

Hi Nick,


I guessed the machine stamp from MRC/CCP4 format description - half byte 01 
means BE, half byte 04 means LE. Are these only applicable to intel machines? 
How are other machine architectures indicated? I do not know. We probably can 
find the authoritative answer from the CCP4 library, just need a little bit 
more time...


The machine stamp itself should not be affected by the machine's architecture, 
because it needs to be read before the program knows what the architecture is. 
Therefore it should be a string instead of a number. In the MRC/CCP4 map 
specification, it says that only the first two bytes are used.  I had seen some 
home-brew programs taking shortcuts by using the int value of the machine stamp 
word, and I thought that was smart. Now I realise that this practice has the 
risk of failing on non-intel machines. So, if it is meant to be half-bytes, 
interpret it as half bytes!


Zhijie



From: Nicholas Devenish 
Sent: Wednesday, November 14, 2018 8:54 AM
To: Zhijie Li
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] VERY old mtz file..

Hi Zhijie,

Looks like we both had the same thoughts!

On Wed, Nov 14, 2018 at 1:19 PM Zhijie Li 
mailto:zhijie...@utoronto.ca>> wrote:

The semiBE.mtz has the big endian stamp (0x11 11 00 00 at bytes 9-12 ) put 
in the header of the original file; everything else is untouched

Is this correct? I thought machine stamp was supposed to be the 4-bit sizes of 
int, real, complex and char e.g. all the little endian MTZ's I've seen have 
0x44 41 00 00, which would make the big-endian version 0x00 00 41 44. Then 
again, mtzdmp complained regardless, and perhaps I've just misinterpreted the 
documentation (I'd love to know if this was the case!)

Thanks,

Nick





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Re: [ccp4bb] VERY old mtz file..

[Zhijie Li]

Hi Nick,

Our LE outputs are exactly the same. Rmerge=100.0%!

Zhijie



From: CCP4 bulletin board  on behalf of Nicholas 
Devenish 
Sent: Wednesday, November 14, 2018 7:15 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] VERY old mtz file..

Hi Eleanor,

On Wed, Nov 14, 2018 at 10:27 AM Eleanor Dodson 
<176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>
 wrote:
Here is the file I was trying to read - please feel free to play with it!!
Eleanor

This is an interesting file - it diverges from several expectations of what I 
thought an MTZ had to look like (by the admittedly sparse documentation I can 
find). Specifically:
- It's big-endian (this should fine)
- The machine stamp - which should identify it as big-endian, is missing 
(interestingly, mtzdmp doesn't appear to use this indicator - I set it to the 
big-endian version and it just complained that it was corrupted)
- Was apparently written before MTZ datasets were a thing, COLSRC property I 
also didn't know was optional...

The good news is that I swapped the endian-ness of the binary parts and wrote 
the machine stamp, and mtzdmp appears to parse the file now. Not really sure 
what other tools you'd need to verify but this might be enough. I've attached.

Thanks,

Nick




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Re: [ccp4bb] VERY old mtz file..

[Zhijie Li]

It's also said here, at the end of file :

https://www.cs.auckland.ac.nz/~patrice/210LN/DR4.pdf

"add 1 to the left, with the binary point"

0.1.




From: CCP4 bulletin board  on behalf of Zhijie Li 

Sent: Tuesday, November 13, 2018 7:43 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] VERY old mtz file..


Hi all,


I think I know why it is a division of 4 instead of 2 is involved in conversion 
from VAX to IEEE now. Short answer: a 2 is in the exponent bits (bias of 128 
instead of 127, visible), another 2 is hidden in the scientific notation.


I found this explanation+example on VAX F-float:

http://courseweb.stthomas.edu/tpsturm/private/notes/qm300/FLOATPT.html


So for IEEE754 float32, if we want to represent a same 12.75 (1100.11) in the 
above example, we  would first conceptually write it in scientific notation as 
1.10011 x 1000 in binary. Then the mantissa part is the part after the dot 
filled with zero to 23 bits: '1001100', the exponent part is 
3+127=130 (dec)=1010(bin). Then the binary IEEE754 float32 number is 
0[1010][1001100]. (You can check it here: 
https://www.h-schmidt.net/FloatConverter/IEEE754.html)


Now compare this with the VAX 12.75 in the linked example, we can find that 
besides the bias becoming 128, the conceptual binary scientific notation is 
actually  0.110011 x 1, instead of 1.10011 x 1000.  So the exponent needed 
is 4 instead of 3. Then the exponent bits are 4+128=132=1100 and the VAX 
float32 becomes

0[1100][1001100] ---if we write in a IEEE-style order. Note 
that the mantissa appears to be same as the ieee mantissa, and the exponent to 
be applied is 132-128=4. If this number is interpreted as IEEE754, then it will 
be 1.10011 x 2exp(132-127)=1.10011 x 10, four times of what it should be.


So, for normalised values, rearranging the VAX F-float bytes, reading as IEEE, 
then dividing by 4 gives the correct value. (The C[0]-1 treatment in the ccp4 
lib is neat.)


In this link describing VAX floats, it is unfortunate that it only states that 
the bias for F-float is 128, but not that the mantissa starts from 0.01 instead 
of 0.1. Therefore the confusion.

https://nssdc.gsfc.nasa.gov/nssdc/formats/VAXFloatingPoint.htm



Thanks to all responded!


Zhijie



From: Ian Tickle 
Sent: Tuesday, November 13, 2018 4:54 PM
To: Zhijie Li
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] VERY old mtz file..


Hi Zhijie

It's definitely a factor 4.  The code is in subroutine QTIEEE in the Fortran 
source I mentioned previously at this line:

See line:

  A(I)=((A(I)+SIGN(2,A(I)))/4.AND..NOT.MNAN).OR.MDN2

If you prefer it in C code it's in function vaxF2ieeeF in:

ccp4-7.0-src/checkout/libccp4/ccp4/vmslibrary.c

See line:

out.c[0] = buffer[i].c[1] - (uint8)1; /* subtracts 2 from exponent */

i.e. subtract 2 from exponent -> division by 4.

Cheers

-- Ian


On Tue, 13 Nov 2018 at 19:52, Zhijie Li 
mailto:zhijie...@utoronto.ca>> wrote:
If somebody is going to send these files by email, please send one to me too. 
Thanks in advance. I actually prefer to get a MTZ file because the miller 
indices would serve as good clues for understanding the encodings.  Even the 
first 1024 bytes of an MTZ would do (data array starts at byte 80 in MTZ).

In my life I had only seen ieee754.  According to what I can find, VAX has an 
exponent bias of 128 (ieee754 uses 127). Then it seems to me that when 
converting from vax to ieee a division of 2 is involved. However all procedures 
I have seen use a division of 4, which is quite puzzling to me. A real data 
file containing meaningful numbers (eg., HKL indices) would be very helpful. 
Thanks in advance.

Zhijie

> On Nov 13, 2018, at 2:21 PM, Johan Hattne 
> mailto:hat...@ucla.edu>> wrote:
>
> Related by not exactly on topic: would anybody on the list be able to share 
> old map files (not MTZ:s) with Convex, Cray, Fujitsu, or VAX reals/strings?  
> I’d be interested to see what those files actually look(ed) like.
>
> // Best wishes; Johan
>
>> On Nov 9, 2018, at 18:38, Zhijie Li 
>> mailto:zhijie...@utoronto.ca>> wrote:
>>
>> Hi all,
>>
>> On linux there are a few good GUI HEX editors. Here I’d like to recommend 
>> BLESS, which conveniently displays all possible numerical interpretations of 
>> the four bytes under cursor. It also allows the user to switch between big 
>> endian or little endian through a checkbox. Unfortunately all floats are 
>> assumed to be IEEE754, therefore VAX floats won’t be interpreted correctly.  
>> ( The simplest way to convert vax to ieee float would be to write a little 
>> program to do some bit operations. I’d be happy to take that as my weekend 
>> project)
>>
>>
>> BTW, along the line of space efficiency, I can

Re: [ccp4bb] VERY old mtz file..

[Zhijie Li]

Hi all,


I think I know why it is a division of 4 instead of 2 is involved in conversion 
from VAX to IEEE now. Short answer: a 2 is in the exponent bits (bias of 128 
instead of 127, visible), another 2 is hidden in the scientific notation.


I found this explanation+example on VAX F-float:

http://courseweb.stthomas.edu/tpsturm/private/notes/qm300/FLOATPT.html


So for IEEE754 float32, if we want to represent a same 12.75 (1100.11) in the 
above example, we  would first conceptually write it in scientific notation as 
1.10011 x 1000 in binary. Then the mantissa part is the part after the dot 
filled with zero to 23 bits: '1001100', the exponent part is 
3+127=130 (dec)=1010(bin). Then the binary IEEE754 float32 number is 
0[1010][1001100]. (You can check it here: 
https://www.h-schmidt.net/FloatConverter/IEEE754.html)


Now compare this with the VAX 12.75 in the linked example, we can find that 
besides the bias becoming 128, the conceptual binary scientific notation is 
actually  0.110011 x 1, instead of 1.10011 x 1000.  So the exponent needed 
is 4 instead of 3. Then the exponent bits are 4+128=132=1100 and the VAX 
float32 becomes

0[1100][1001100] ---if we write in a IEEE-style order. Note 
that the mantissa appears to be same as the ieee mantissa, and the exponent to 
be applied is 132-128=4. If this number is interpreted as IEEE754, then it will 
be 1.10011 x 2exp(132-127)=1.10011 x 10, four times of what it should be.


So, for normalised values, rearranging the VAX F-float bytes, reading as IEEE, 
then dividing by 4 gives the correct value. (The C[0]-1 treatment in the ccp4 
lib is neat.)


In this link describing VAX floats, it is unfortunate that it only states that 
the bias for F-float is 128, but not that the mantissa starts from 0.01 instead 
of 0.1. Therefore the confusion.

https://nssdc.gsfc.nasa.gov/nssdc/formats/VAXFloatingPoint.htm



Thanks to all responded!


Zhijie



From: Ian Tickle 
Sent: Tuesday, November 13, 2018 4:54 PM
To: Zhijie Li
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] VERY old mtz file..


Hi Zhijie

It's definitely a factor 4.  The code is in subroutine QTIEEE in the Fortran 
source I mentioned previously at this line:

See line:

  A(I)=((A(I)+SIGN(2,A(I)))/4.AND..NOT.MNAN).OR.MDN2

If you prefer it in C code it's in function vaxF2ieeeF in:

ccp4-7.0-src/checkout/libccp4/ccp4/vmslibrary.c

See line:

out.c[0] = buffer[i].c[1] - (uint8)1; /* subtracts 2 from exponent */

i.e. subtract 2 from exponent -> division by 4.

Cheers

-- Ian


On Tue, 13 Nov 2018 at 19:52, Zhijie Li 
mailto:zhijie...@utoronto.ca>> wrote:
If somebody is going to send these files by email, please send one to me too. 
Thanks in advance. I actually prefer to get a MTZ file because the miller 
indices would serve as good clues for understanding the encodings.  Even the 
first 1024 bytes of an MTZ would do (data array starts at byte 80 in MTZ).

In my life I had only seen ieee754.  According to what I can find, VAX has an 
exponent bias of 128 (ieee754 uses 127). Then it seems to me that when 
converting from vax to ieee a division of 2 is involved. However all procedures 
I have seen use a division of 4, which is quite puzzling to me. A real data 
file containing meaningful numbers (eg., HKL indices) would be very helpful. 
Thanks in advance.

Zhijie

> On Nov 13, 2018, at 2:21 PM, Johan Hattne 
> mailto:hat...@ucla.edu>> wrote:
>
> Related by not exactly on topic: would anybody on the list be able to share 
> old map files (not MTZ:s) with Convex, Cray, Fujitsu, or VAX reals/strings?  
> I’d be interested to see what those files actually look(ed) like.
>
> // Best wishes; Johan
>
>> On Nov 9, 2018, at 18:38, Zhijie Li 
>> mailto:zhijie...@utoronto.ca>> wrote:
>>
>> Hi all,
>>
>> On linux there are a few good GUI HEX editors. Here I’d like to recommend 
>> BLESS, which conveniently displays all possible numerical interpretations of 
>> the four bytes under cursor. It also allows the user to switch between big 
>> endian or little endian through a checkbox. Unfortunately all floats are 
>> assumed to be IEEE754, therefore VAX floats won’t be interpreted correctly.  
>> ( The simplest way to convert vax to ieee float would be to write a little 
>> program to do some bit operations. I’d be happy to take that as my weekend 
>> project)
>>
>>
>> BTW, along the line of space efficiency, I can’t help noticing that the 
>> miller indices are saved as float32 in mtz, as all other numbers in mtz. 
>> This certainly have made mtz format a beautiful homogeneous data format ;).  
>> In this particular case, if we have doubts about the reliability of the 
>> machine stamp, trying to restore the miller indices would be a good way to 

Re: [ccp4bb] VERY old mtz file..

[Zhijie Li]

Hi Ethan,
Thanks for the information. My guess is that in MTZ only F-float is expected, 
because it is the only 32bit form? 
Zhijie

> On Nov 13, 2018, at 3:44 PM, Ethan A Merritt  wrote:
> 
>> On Tuesday, November 13, 2018 11:51:55 AM PST Zhijie Li wrote:
>> If somebody is going to send these files by email, please send one to me 
>> too. Thanks in advance. I actually prefer to get a MTZ file because the 
>> miller indices would serve as good clues for understanding the encodings.  
>> Even the first 1024 bytes of an MTZ would do (data array starts at byte 80 
>> in MTZ).
>> 
>> In my life I had only seen ieee754.  According to what I can find, VAX has 
>> an exponent bias of 128 (ieee754 uses 127). Then it seems to me that when 
>> converting from vax to ieee a division of 2 is involved.
> 
> It's more complicated than that.  VAXen supported multiple floating point 
> formats,
> F-floating G-floating and H-floating.
> They had differed by how many bits were used for the exponent, and hence how
> many bits were left for the mantissa.
> I can pull out the architecture manuals if necessary.
> 
>ah, nostalgia
> 
>Ethan
> 
> 
>> However all procedures I have seen use a division of 4, which is quite 
>> puzzling to me. A real data file containing meaningful numbers (eg., HKL 
>> indices) would be very helpful. Thanks in advance.
>> 
>> Zhijie
>> 
>>> On Nov 13, 2018, at 2:21 PM, Johan Hattne  wrote:
>>> 
>>> Related by not exactly on topic: would anybody on the list be able to share 
>>> old map files (not MTZ:s) with Convex, Cray, Fujitsu, or VAX reals/strings? 
>>>  I’d be interested to see what those files actually look(ed) like.
>>> 
>>> // Best wishes; Johan
>>> 
>>>> On Nov 9, 2018, at 18:38, Zhijie Li  wrote:
>>>> 
>>>> Hi all,
>>>> 
>>>> On linux there are a few good GUI HEX editors. Here I’d like to recommend 
>>>> BLESS, which conveniently displays all possible numerical interpretations 
>>>> of the four bytes under cursor. It also allows the user to switch between 
>>>> big endian or little endian through a checkbox. Unfortunately all floats 
>>>> are assumed to be IEEE754, therefore VAX floats won’t be interpreted 
>>>> correctly.  ( The simplest way to convert vax to ieee float would be to 
>>>> write a little program to do some bit operations. I’d be happy to take 
>>>> that as my weekend project)
>>>> 
>>>> 
>>>> BTW, along the line of space efficiency, I can’t help noticing that the 
>>>> miller indices are saved as float32 in mtz, as all other numbers in mtz. 
>>>> This certainly have made mtz format a beautiful homogeneous data format 
>>>> ;).  In this particular case, if we have doubts about the reliability of 
>>>> the machine stamp, trying to restore the miller indices would be a good 
>>>> way to test hypotheses.
>>>> 
>>>> Zhijie
>>>> 
>>>>> On Nov 9, 2018, at 9:04 PM, James Holton 
>>>>> <270165b9f4cf-dmarc-requ...@jiscmail.ac.uk> wrote:
>>>>> 
>>>>> As a beamline scientist I must say I am glad that diffraction image data 
>>>>> is not usually stored as ASCII text.  In fact, I am slowly warming to the 
>>>>> idea of storing it as not just binary, but compressed formats.  Problem, 
>>>>> I'm sure will be that it won't be  long before we forget how to 
>>>>> decompress them, as most of the algorithms we are using aren't all that 
>>>>> widespread.  Probably around the same time future generations will curse 
>>>>> us for using ASCII instead of unicode, which is a 16-bit standard. I'm 
>>>>> sure we will be reviled for limiting ourselves so, just to save a factor 
>>>>> of two in disk space.
>>>>> In situations like this I always use the unix "od" command.  It makes 
>>>>> everything "human readable" by converting the bytes into strings you can 
>>>>> read.  Then it is just a matter of figuring out what the bytes are.
>>>>> Unfortunately, "od" only decodes floats on the native platform, so if the 
>>>>> mtz is from another platform (Windows vs Linux, for example), then you 
>>>>> might need to do some swapping.  Thus far, I have encountered files that 
>>>>> require one of a few swapping strategies in order to make them work:
>>>>> 
>>>>> 1 2 3 4 -

Re: [ccp4bb] VERY old mtz file..

[Zhijie Li]

If somebody is going to send these files by email, please send one to me too. 
Thanks in advance. I actually prefer to get a MTZ file because the miller 
indices would serve as good clues for understanding the encodings.  Even the 
first 1024 bytes of an MTZ would do (data array starts at byte 80 in MTZ).

In my life I had only seen ieee754.  According to what I can find, VAX has an 
exponent bias of 128 (ieee754 uses 127). Then it seems to me that when 
converting from vax to ieee a division of 2 is involved. However all procedures 
I have seen use a division of 4, which is quite puzzling to me. A real data 
file containing meaningful numbers (eg., HKL indices) would be very helpful. 
Thanks in advance.

Zhijie

> On Nov 13, 2018, at 2:21 PM, Johan Hattne  wrote:
> 
> Related by not exactly on topic: would anybody on the list be able to share 
> old map files (not MTZ:s) with Convex, Cray, Fujitsu, or VAX reals/strings?  
> I’d be interested to see what those files actually look(ed) like.
> 
> // Best wishes; Johan
> 
>> On Nov 9, 2018, at 18:38, Zhijie Li  wrote:
>> 
>> Hi all,
>> 
>> On linux there are a few good GUI HEX editors. Here I’d like to recommend 
>> BLESS, which conveniently displays all possible numerical interpretations of 
>> the four bytes under cursor. It also allows the user to switch between big 
>> endian or little endian through a checkbox. Unfortunately all floats are 
>> assumed to be IEEE754, therefore VAX floats won’t be interpreted correctly.  
>> ( The simplest way to convert vax to ieee float would be to write a little 
>> program to do some bit operations. I’d be happy to take that as my weekend 
>> project)
>> 
>> 
>> BTW, along the line of space efficiency, I can’t help noticing that the 
>> miller indices are saved as float32 in mtz, as all other numbers in mtz. 
>> This certainly have made mtz format a beautiful homogeneous data format ;).  
>> In this particular case, if we have doubts about the reliability of the 
>> machine stamp, trying to restore the miller indices would be a good way to 
>> test hypotheses.
>> 
>> Zhijie
>> 
>>> On Nov 9, 2018, at 9:04 PM, James Holton 
>>> <270165b9f4cf-dmarc-requ...@jiscmail.ac.uk> wrote:
>>> 
>>> As a beamline scientist I must say I am glad that diffraction image data is 
>>> not usually stored as ASCII text.  In fact, I am slowly warming to the idea 
>>> of storing it as not just binary, but compressed formats.  Problem, I'm 
>>> sure will be that it won't be  long before we forget how to decompress 
>>> them, as most of the algorithms we are using aren't all that widespread.  
>>> Probably around the same time future generations will curse us for using 
>>> ASCII instead of unicode, which is a 16-bit standard. I'm sure we will be 
>>> reviled for limiting ourselves so, just to save a factor of two in disk 
>>> space.
>>> In situations like this I always use the unix "od" command.  It makes 
>>> everything "human readable" by converting the bytes into strings you can 
>>> read.  Then it is just a matter of figuring out what the bytes are.
>>> Unfortunately, "od" only decodes floats on the native platform, so if the 
>>> mtz is from another platform (Windows vs Linux, for example), then you 
>>> might need to do some swapping.  Thus far, I have encountered files that 
>>> require one of a few swapping strategies in order to make them work:
>>> 
>>> 1 2 3 4 - no swapping
>>> 
>>> 4 3 2 1 - reverse all bytes
>>> 
>>> 3 4 1 2 - swap words and swap bytes within the words
>>> 2 1 4 3 - reverse of previous
>>> 
>>> 2-1 1 4 3 - same as last, but if not all zero, decrement byte #2 before 
>>> swapping
>>> 3 4 1 2+1 - same as 3412, but if not all zero increment byte #2 before 
>>> swapping
>>> I'm sure there are other combinations, but the oldest MTZ I have is only 
>>> from 1996.
>>> 
>>> -James Holton
>>> MAD Scientist
>>> 
>>> 
>>>> On 11/9/2018 4:47 AM, Eleanor Dodson wrote:
>>>> Anyone any idea what to do about this?? Created in 1992!!
>>>> Seems unreadable..
>>>> 
>>>> No CTYP lines input for file:  1
>>>>Indices output even if all data items flagged "missing"
>>>> Warning, NOT all LABOUT data lines given
>>>> Warning: Machine stamp corrupted? Assuming native format. 
>>>>>>>>>> CC

Re: [ccp4bb] VERY old mtz file..

[Zhijie Li]

Hi all,

On linux there are a few good GUI HEX editors. Here I’d like to recommend 
BLESS, which conveniently displays all possible numerical interpretations of 
the four bytes under cursor. It also allows the user to switch between big 
endian or little endian through a checkbox. Unfortunately all floats are 
assumed to be IEEE754, therefore VAX floats won’t be interpreted correctly.  ( 
The simplest way to convert vax to ieee float would be to write a little 
program to do some bit operations. I’d be happy to take that as my weekend 
project)


BTW, along the line of space efficiency, I can’t help noticing that the miller 
indices are saved as float32 in mtz, as all other numbers in mtz. This 
certainly have made mtz format a beautiful homogeneous data format ;).  In this 
particular case, if we have doubts about the reliability of the machine stamp, 
trying to restore the miller indices would be a good way to test hypotheses.

Zhijie

On Nov 9, 2018, at 9:04 PM, James Holton 
<270165b9f4cf-dmarc-requ...@jiscmail.ac.uk>
 wrote:


As a beamline scientist I must say I am glad that diffraction image data is not 
usually stored as ASCII text.  In fact, I am slowly warming to the idea of 
storing it as not just binary, but compressed formats.  Problem, I'm sure will 
be that it won't be long before we forget how to decompress them, as most of 
the algorithms we are using aren't all that widespread.  Probably around the 
same time future generations will curse us for using ASCII instead of unicode, 
which is a 16-bit standard. I'm sure we will be reviled for limiting ourselves 
so, just to save a factor of two in disk space.

In situations like this I always use the unix "od" command.  It makes 
everything "human readable" by converting the bytes into strings you can read.  
Then it is just a matter of figuring out what the bytes are.

Unfortunately, "od" only decodes floats on the native platform, so if the mtz 
is from another platform (Windows vs Linux, for example), then you might need 
to do some swapping.  Thus far, I have encountered files that require one of a 
few swapping strategies in order to make them work:

1 2 3 4 - no swapping

4 3 2 1 - reverse all bytes

3 4 1 2 - swap words and swap bytes within the words

2 1 4 3 - reverse of previous

2-1 1 4 3 - same as last, but if not all zero, decrement byte #2 before swapping

3 4 1 2+1 - same as 3412, but if not all zero increment byte #2 before swapping

I'm sure there are other combinations, but the oldest MTZ I have is only from 
1996.

-James Holton
MAD Scientist


On 11/9/2018 4:47 AM, Eleanor Dodson wrote:
Anyone any idea what to do about this?? Created in 1992!!
Seems unreadable..

No CTYP lines input for file:  1
Indices output even if all data items flagged "missing"
 Warning, NOT all LABOUT data lines given
Warning: Machine stamp corrupted? Assuming native format.
>> CCP4 library signal library_file:End of File (Error)




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Re: [ccp4bb] [Offtopic] Protein stain in Coomassie Blue dyes

[Zhijie Li]

Hi,

At high concentration (1-2%) the published saturating SDS:protein binding ratio 
is about 1.4:1 by weight, that is roughly one SDS molecule per two aa on 
average. It is dense but  not that dense to prevent any further interaction.  
More importantly, as a quite hydrophilic small molecule SDS should have no 
trouble dissociating from the peptide when its in-solution concentration drops 
(therefore you can use SDS gel bands for MS). With common procedure, during 
staining the SDS should partially fall off( especially if the gel is heated), 
and partially remain with the protein in the gel, depending on: how hydrophobic 
the protein is, how low the environmental SDS concentration becomes, how much 
organic solvent there is in the solution, etc.. The coomassie should simply 
find whatever hydrophobic/positively charged patch to bind and aggregate. 
Besides, coomassie-R is probably slightly more hydrophobic than SDS so it is 
capable of competing SDS off if necessary. (The even less hydrophobic Coomassie 
G250 definitely binds protein in the presence of detergents - that how Blue 
Native gel works for membrane proteins) Finally, since the only thing you are 
looking for is some deeper blue to indicate the presence of protein, even if 
SDS did prevent dye binding to some extent, your gel still will work. This is 
different from when you want to use the dye to do some quantitative work such 
as the Bradford assay. It would be interesting to know the effect of detergents 
on Bradford.

Zhijie



On Oct 4, 2018, at 9:26 PM, Alex Lee 
mailto:alexlee198...@gmail.com>> wrote:

Dear All,

I am thinking that in an SDS-PAGE experiment, if protein samples are boiled in 
SDS containing loading dye, and supposedly SDS interacts with proteins, why the 
Coomassie Blue dyes could still interact with and stain the proteins?  I am 
thinking SDS is covering the proteins, making no room for the Coomassie Blue 
dyes interaction.  I'd appreciate it if any input from this forum.

Alex



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Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Electron density maps for Cryo-EM structures.

[Zhijie Li]

Hi Kevin,

a)

If your goal is merely to display EM maps, then UCSF Chimera, COOT, 
pymol, etc. should all do. The EM maps are saved in the MRC format (.mrc 
or .mrcs). Despite a different extension and some minor differences in 
the headers, the MRC format is essentially the same format as our 
electron density maps (.ccp4 .map, etc.). Your favorite software for 
displaying crystallographic maps should work just fine. You will later 
find that owing to the fact that images are just 2D pixels, movies are 
series of 2D pixels,  maps are (saved as) 3D voxels, the single MRC/CCP4 
format can hold many different types of data, including images, stacks 
of images, movies, maps, masks, etc..  That is why there is often also a 
.star file (similar to CIF files) that holds information on what is 
inside one or more MRC files. So be aware of what type of data you are 
opening.


b)

To get an idea how cryo-EM single particle data processing works, the 
minimum you need would be RELION/EMAN2+UCSF Chimera (best for this job), 
which are all free to everyone. These will get you from the raw EM 
movies to the EM maps.


If you have a computer with enough memory (16GB minimum, the more the 
better) and an Nvidia 1080TI card (~USD 800? a $150 1050TI can also get 
you started) you can already solve some EM structures! To do this, you 
need to get RELION and/or EMAN2 (ideally both):


https://www2.mrc-lmb.cam.ac.uk/relion/index.php?title=Download_%26_install

http://blake.bcm.edu/emanwiki/EMAN2/Install

The reason for the 1080TI card is that it allows the programs to use its 
300+ GPU cores to accelerate computations. This capability is provided 
by Nvidia's CUDA library. You need to download the CUDA 8.0 library from 
Nvidia. CUDA 8.0 needs to be installed before you compile RELION if you 
are going to compile it yourself (not quite necessary).


At present, EMAN2 only uses GPU at the particle picking step so it is 
not essential to have a GPU card just for running EMAN2. But 
classifications and refinements will be very slow (days and weeks) 
without a GPU.


Ideally you should install everything in Linux, such as Ubuntu 16.04 
mate. You will also need softwares such as Motioncor2, GCTF, CTFFind, 
for some jobs in the workflow. Certain settings need to be put as 
environmental variables in the Linux system. Please figure them out 
yourself. It will take days to succeed for first-timers.



You can start playing by following the tutorials of either RELION or 
EMAN2 with their own tutorial datasets. These datasets are not really 
raw data though: they are particle "stacks" that contain the particles 
picked from the raw micrographs. But starting from there would be the 
easiest way to learn the essentials. BTW, following the EMAN2 tutorial 
does not involve using a GPU at all. This might be true for the non-GPU 
version of RELION too.



If you want to start from the very beginning, you can download the 396GB 
proteasome movie dataset from EMPIAR:


https://www.ebi.ac.uk/pdbe/emdb/empiar/entry/10025/


On youtube, Grant Jensen has a great series on cryoEM basics. I strongly 
suggest you to watch at least the part1, especially those having to do 
with CTF.


https://www.youtube.com/watch?v=gDgFbAqdM_c=PL8_xPU5epJdctoHdQjpfHmd_z9WvGxK8-

Zhijie




On 10/09/2018 1:01 PM, Kevin Jin wrote:

Dear Herman and all ccp4ers,

Many thanks for the helps and education from all of you.

I am a fresh beginner in Cryo-EM,  and have no access to those 
resource, like Chimera, Phenix, Rosetta and papers, etc. For me, any 
answer will be valuable.


Please forgive me for asking such a naive question.  I highly 
appreciate your comments and education.


Kevin

On Mon, Sep 10, 2018 at 5:28 AM > wrote:


Hi Kevin, Pavel and others,

Since it seems that so far nobody answered the primary question:
“Is there any sever available to create electron density maps for
cryo-em structures?” So I will do it. The answer is very simple:
They do not need to be created, they are available from the pdb!

To give an example: On the RCSB pdb web-site, I searched for entry
5vai. Then under the button “Download Files” I selected “Download
EM Map” and downloaded emd_8653.map.gz. As the name suggests, this
file needs to be unzipped, but this is trivial.

Then in Coot in the “File” pull-down menu, I select “Open Map” to
load the map.

Next, you may not see anything since to contour level might be too
high (when I load the map, the contour level is around 6 rmsd) so
you have to scroll down the contour level to see anything. As
Marin and Ian pointed out, the maps are very similar to regular
electron density maps, probably with the exception that the EM
“electron density” maps are influenced by the local charge
density. However in coot they behave 100% identical and you can
scroll up and down the contour level as you like.

Of course, if the authors did 

Re: [ccp4bb] Off topic: denaturing urea gels

[Zhijie Li]

Smith:
One should expect to see ladders each separated by 1 nt in such cases.

Mohammed:

My suggestions:
1) running at 70V seems low. I do not see a reason for not using higher 
voltages. 200Vx45min should be OK. IT is better to not let the BPB front run 
out of the gel - so that you know whether the DNA had all been chopped into 
single nts. (how do you visualize the bands? Is the oligo end-labelled?  EtBr 
should not work well for small fragments. But UV shadowing may work.)

2) boil the sample before loading (6-8 M urea should always be included in 
sample buffers). This is important because the enzyme might bind the DNA and 
slowly release them causing the smearing.

3) How does the uncleaved DNA alone (no enzyme at all) look on the same gel? If 
it is a sharp single band then you might need to pay extra attention to 
denaturing the samples before loading. If it is also smeary then it might be 
the gel system needs optimizing.

4) use 0.75mm gel. Thinner gels give better resolution. 

5) I found that DNA ran just fine in the discontinuous tris-glycine 
system(Laemmli). In other words,  the PAGE system routinely used for proteins 
(without SDS) works for DNA too, with the benefit of sharpening the bands.
 You may also consider experimenting with gel percentages(25%-30 % for example) 
so that you can see even single nts. The acry:bis ratio is not that important 
as long as you stick to one.


Zhijie



> On Sep 30, 2017, at 7:54 AM, Smith Liu  wrote:
> 
> your enzyme cannot give definite fragment. thus smear
> 
> 
> 发自网易邮箱大师
> 
> 在2017年09月30日 19:28，Mohammad Khan 写道：
> Dear all,
> 
> I am working with an exonuclease and I run the digested DNA on a 
> 8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system 
> and cast gels of about 8.6x6.5 cm dimensions with 1.5 mm thickness. I use a 
> 15 well comb. I run my gels at 70 V for as long as 4 hours till my undigested 
> DNA reaches half the gel distance. I use 20-30 nt long susbtrates.
> 
> I am mostly not able to get distinct bands of the digested products but 
> rather get a smear. Is there any way to make sure that I get distinct 
> digested products rather than a smear?
> 
> I am looking forward for suggestions from all!
> 
> Thank you.
> 
> Ciao!

Re: [ccp4bb] Best method for carbohydrate refinement

[Zhijie Li]

Hi Gustavo,

If I understand you correctly, you are concerned about N-glycans 
(N-glycosylation) on your proteins. According to your description, you 
have 2 protein molecules in each ASU, each bearing one potential 
N-glycan site. Then there are only two N-glycan sites you need to build 
for each dataset ( I suppose you are not going to deposit everyone of 
them? ). In most cases we do not see much ordered part of the N-glycans, 
usually just one or both of the core GlcNac (NAG) residues . If this is 
the case then it is not much work.


In very lucky cases you may see one or two rather complete N-glycans. I 
think insect cells produce mostly pauci-mannose N-glycans (mostly 
Man3-GlcNac2, less frequently Man4-GlcNac2) possibly carrying 
core-fucosylation on the innermost GlcNAc.  Note that although mammals 
only have core 1,6 fucose, insects may also have an additional core 1,3 
fucose, discussed and illustrated in the following papers:


https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3589692/

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3647355/

I am not aware of an automated procedure for building complex glycans 
such as the N-glycans, so take extreme care making the correct linkages 
if you have to build more than the two core NAGs. The Man3Gn2-Asn should 
be 
Man_alpha1,3-[Man_alpha1,6]-Man_beta1,4-GlcNac_beta1,4-GlcNAc_beta-Asn. 
The core fucose(s), if present, will be alpha1,6- or alpha1,3- linked to 
the innermost GlcNAc. Note that the core alpha1,3 fucose modification 
occurs only when the core alpha1,6 is already there.



Zhijie


On 28/07/2017 1:43 PM, Gustavo Machado Alvares De Lima wrote:

Hello everybody,

I was looking for suggestion on the best way to identify and refine 
carbohydrates in a protein. This is the scenario:

- I have 10 molecules in the biological assembly
- I used sf9 expression system, so I have random glycolisation and types of 
glycolisations. I have some hint about possible glycolisation sites
- I believe I have only 1 glysolisation per monomer
- I have 2 molecule per AU. Space group C2


I would like (even if I have to write down a script for it) to track this 
glycolisation, identify the most probable ones, determine the restrictions for 
each and refine them. I could do it by hand on every refinement cycle (or a 
couple of cycles), but for many datasets it would take ages.

What protocol would you suggest?

I really appreciate any help in this subject.

Regards,
Gustavo

Re: [ccp4bb] NCS axis detrmination

[Zhijie Li]

Hi Vandna,

Assuming you have two copies, chain A and B.

In UCSF chimera:

1) open two copies of the pdb (model #0 and #1)
2) in command line, type:

match   #1:.B@CA   #0:.A@CA showMatrix true

Besides moving model #1 chain B to model #0 chain A, the match command 
also outputs the rotation and translation matrices and angles, axes in 
Favorite-> Reply Log.


Note that you might need to specify residue number range for the two 
chains if the number of CAs in them are not equal - just to make the 
match working. The match command does not guess the range of residues to 
align as the MatchMaker does. Alt confs involving CAs also count. You 
may run:


del @CA.B

to delete all alt conf B CA atoms before the match command to make it 
working



If you wish to draw the rotation axis in Chimera, you need to make a 
bild file  that draws an arrow from the "Axis point" with direction 
specified by the Axis vector. Assuming the "Axis" is "Ax Ay Az" and the 
"Axis point" is "x y z", you need to calculate X1=x+Ax*20,  Y1=y+Ay*20, 
Z1=z+Az*20, then put this  in the arrow.bld file:


.arrow x y z X1 Y1 Z1

https://www.cgl.ucsf.edu/chimera/docs/UsersGuide/bild.html


COOT also outputs the matrices in the terminal window. But COOT does not 
seem to directly show the more human friendly "rotation angle" and 
"shift along axis".


Zhijie



On 29/06/2017 3:16 PM, Vands wrote:

Hi  every one ,
I am looking for a tool to perfectly determine 
the NCS axis. I have two forms of crystals where diamer is with NCS 
but on a comparison of both forms, it looks like NCS axis is little 
off when i superpose two forms, i would like to measure rotation or 
translation between axes .



--
Vandna Kukshal
Senior Scientist
Dept. Biochemistry and Molecular Biophysics
Washington University School of Medicine
660 S. Euclid, Campus Box 8231
St. Louis, MO 63110

Re: [ccp4bb] off topic: high resolution animation in chimera

[Zhijie Li]

Update: to get transparent background in GIF:

Chimera command file (now saves PNG with transparent background by 
adding "set bgTransparency"):



close all;open #0 ABCD.pdb;
set bgTransparency;
~disp;ribbon :.a;
movie record directory "D:\mov_temp\"  format png pattern frame_*;
turn y 1 15;wait;turn y -1 15;wait;freeze;
movie stop;

ImageMagick command ("-dispose background" ensures that one frame is 
displayed at a time):


D:\>magickconvert-delay 10   -dispose background -loop 0   
d:\mov_temp\frame_*.png  d:\mov_temp\animation.gif






On 25/05/2017 5:18 PM, Shanti Pal Gangwar wrote:

Dear all,

I am trying to make an animation using chimera. But it looks like that 
the animation output is poor quality, seems less dpi. How to get a 
high resolution animation, say 300 dpi, using ucsf chimera. I am using 
movie record commands.



Thank you very much,


Shanti Pal

Re: [ccp4bb] off topic: high resolution animation in chimera

[Zhijie Li]

Hi Shanti,

To get more control, you can use Chimera to generate the frames, save 
each of them as PNG, then process and re-assemble the frames in 
photoshop to make GIFs.  The command line toolbox Imagemagick is also a 
great way for making GIFs (shown below).



Step1  generating the frames

Modify then save the following into a chimera command file and run it in 
chimera:


close all;open #0 ABCD.pdb;
~disp;ribbon :.a;
movie record directory "D:\mov_temp\"  format png pattern frame_*;
turn y 1 15;wait;turn y -1 15;wait;freeze;
movie stop;



Notes:

1) The command "movie reset" resets the file numbering back to 0, 
but also deletes the saved frames.


2) The output image size is exactly the size of the current chimera 3D 
display window. So resizing the window is a very simple and straight 
forward way for adjusting the final GIF size.




Step 2 Combining the PNG (frames) to make GIF using ImageMagick:

First, install Imagemagick: 
https://www.imagemagick.org/script/download.php .  Assuming that no 
further processing of the frames is needed, in windows, open a cmd 
window, type:


magickconvert-delay 10   -loop 0 
d:\mov_temp\frame_*.png   d:\mov_temp\animation.gif


This combines the png files into a GIF with a frame delay of 100 ms (10 
ms * 10) and loops forever (-loop 0).


Imagemagick can also resize, crop, make transparency, set color 
conversion (as PNG is 24bit color while GIF only allows 8bit), etc. .



Zhijie





On 25/05/2017 5:18 PM, Shanti Pal Gangwar wrote:

Dear all,

I am trying to make an animation using chimera. But it looks like that 
the animation output is poor quality, seems less dpi. How to get a 
high resolution animation, say 300 dpi, using ucsf chimera. I am using 
movie record commands.



Thank you very much,


Shanti Pal

Re: [ccp4bb] Glycoprotein expression question

[Zhijie Li]

Hi Bernhard,

I guess you knew all these and is really asking for people's experience, 
but please excuse me to start from the theory: N-glycans in eukaryotes 
are known to be involved in glycoprotein folding in the ER. They allow 
the nascent protein to get into the calnexin/calreticulin cycle in which 
these lectins/chaperones can recruit the disulfide isomerase ERP57. The 
N-glycans also serve as degradation signals in the ERAD pathways, where 
certain structures signify the cells that all efforts had failed on this 
molecule. Of course, for recombinant overproduction of proteins, these 
factors can both be dispensible: the bad proteins, as long as they can 
get secreted, might get preferentially lost during purification or 
crystallization. In fact considering that N-glycans either tether the 
not-so-folded protein to the Cxn/Crt cycle, or direct the terminally ill 
proteins to degradation, and that the quality control of cells could be 
leaky (which I think was exploited in some CFTR-related cases), one may 
even expect that in certain cases not-so-badly folded proteins may get 
to the surface easier if it does not contain N-glycans - pure 
conjectures though. But indeed there are a lot of disulfide-containing 
eukaryotic proteins that are completely not N-glycosylated in certain 
species while fairly well N-glycosylated (to the common extent of ~1 
site /80-100 residues) in many other species (although the surfaces of 
the proteins will be significantly different too). So, it seems that the 
requirement for N-glycan - chaperone - ERAD may not be very strong for 
every protein.


On the other hand, the first GlcNAc in the N-glycan is sometimes found 
to be very closely associated with the amino acid components of 
proteins, even half-inserted into the folded domain (eg., 1HCN, A/NAG94 
), which is a consequence of the co-translational addition of the 
N-glycans. In such case I would expect that the removal of the 
particular N-glycan by mutagenesis may be quite destructive to folding 
in certain cases. I know that there are reports in which people removed 
N-glycans one by one and observed very significant differences among 
sites on secretion/solubility.


So, my view is that many of the N-glycan sites are removable by 
mutagenesis, but certain sites are not. Therefore if one is faced with 
highly N-glycosylated proteins, it is more of a matter of luck or 
thoroughness if he/she starts to explore the combination of mutations. 
To complicate this further, one may also consider trying different ways 
of mutating the sites - besides changing the Asn to Asp, one can also 
consider mutating the S/T at the third position, which is in fact often 
how an N-glycosylation site gets lost among species. There is also the 
space of the Asn mutants to explore. What I did in the past was mutating 
the Asn to Gln, which does not introduce a charge difference.


Zhijie


On 11/04/2017 4:34 PM, Bernhard Rupp wrote:


Hi Fellows,

a humble question for our glyco-expressionists:

I have mutated out the Asns of the N-glycoslation consensus sites for Asp

(Asp simply because the PNGaseF treated protein stays stable so I 
thought that might be a good guess)


and indeed the unglycosilated mutant expresses well and gets secreted 
as planned.


But rumor has it that glycoproteins that are mutated to non-glyc often 
are not processed correctly and


that we had just dumb luck.

May I poll the educated opinion of the erudite here?

Cheers, BR

--

Bernhard Rupp

Crystallographiae Vindicis Militum Ordo

http://www.hofkristallamt.org/

b...@hofkristallamt.org 

+1 925 209 7429

+43 767 571 0536

--

:(){ :|: & };:

--

Re: [ccp4bb] Coot and Pymol through SSH by Xming/PUTTY on a windows client?

[Zhijie Li]

Hi Chen,

I followed instructions on this page and it seems to be working:

http://www.geo.mtu.edu/geoschem/docs/putty_install.html

One thing I think is worth mentioning is that in Putty-connection-SSH-X11, I 
put localhost:0.0 instead of 10.0, as for putty itself, it is requesting from 
Xming for the use of display 0.0.

For COOT, it seems that I have to set Xlaunch in “one window” mode or simply 
run Xming.exe. But still there is some issue with refreshing the screen when 
choosing menu items.

My systems are CentOS 6.6 and Windows 7/Putty0.60/Xming6.9.0.31.

Zhijie



From: Chen Zhao 
Sent: Wednesday, July 01, 2015 7:05 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Coot and Pymol through SSH by Xming/PUTTY on a windows 
client?

Thank you Dale, but when I added  localhost:10.0  to X display location, the 
problem still exist, just without the phrase localhost:10.0 in the warning. 
My X11 forwarding is enabled all the time and all other GUIs work just fine.


And thank you for your clarification on the concept of server and client in 
the X11 word. It makes a lot of sense and I just didn't give it a second 
thought!


Best,

Chen


On Wed, Jul 1, 2015 at 6:47 PM, Dale Tronrud de...@daletronrud.com wrote:

  -BEGIN PGP SIGNED MESSAGE-
  Hash: SHA1


 Both the ssh client and server must be set up with X11Forwarding
  yes.  The message sounds like your local computer is not set up to
  accept X11 tunneling.  (By the way, in the X11 world the remote system
  is the client and your local system the server.)

  Dale Tronrud


  On 7/1/2015 3:40 PM, Chen Zhao wrote:
   Hi all,
  
   Sorry to bother you, but I am trying to fix a long-standing problem
   that I cannot run Coot and Pymol through Xming/PUTTY by SSH
   connection on a windows client. The error messages are pretty
   similar for both: Coot: PuTTY X11 proxy: unable to connect to
   forwarded X server: Network error: Connection refused
   (coot-real:23113): Gtk-WARNING **: cannot open display:
   localhost:10.0 Pymol: PuTTY X11 proxy: unable to connect to
   forwarded X server: Network error: Connection refused freeglut
   (pymol): failed to open display 'localhost:10.0'
  
   Does anybody have some ideas?
  
   Thank you so much, Chen

  -BEGIN PGP SIGNATURE-
  Version: GnuPG v2.0.22 (MingW32)

  iEYEARECAAYFAlWUbgYACgkQU5C0gGfAG10hVgCeLmuE4pHFrapu9biY9nHO/Bpi
  5O8An17UN+hgpr7/6A+mny+XOBfJV/T5
  =iTfz
  -END PGP SIGNATURE-

Re: [ccp4bb] Hi

[Zhijie Li]

Hi Vijay,

I am not sure if I understand you correctly. If you want to align by the two 
nucleotide in two GTPase structures, you can simply do it in COOT using the 
“LSQ superpose” function. You need to fill in the chain id and residue number 
only for the nucleotide and choose “all atoms”. Unless you want to do this for 
more than 10 structures I think using COOT would be the most simple way. For 
more than 10, you can use the CCP4 program LSQKAB to do it in a batch format. 

If the two nucleotides have differences in atom naming or if some atoms are 
missing, you may need to edit one of them (or model a “fresh” one), although I 
guess for GTP/GDP this should not be a frequent problem.

Zhijie



From: vijay srivastava 
Sent: Tuesday, May 26, 2015 8:41 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Hi

Dear All,

I want to superpose the nucleotide form one GTPase on to the nucleotide of 
other GTPase in order to study 

the interaction in the nucleotide binding pocket. I tried to superpose but it 
is superposing on the basis of secondary structure as a
result both the nucleotides from two structutres are not properly aligned.  I 
want to superpose both  the nucleotide, so that I will get the matrix, which I 
want to apply on my desired strcuture and study the interacting residues.
Do any one have the align program with you or any other program which can solve 
this problem.

waiting for your kind response

regards
vijay

Re: [ccp4bb] Superdex vs Sephadex

[Zhijie Li]

Hi Ivan,

In simple words: superdex is a few generations newer than sephadex. Main 
difference: superdex is a physically and chemically, tougher medium.

Among all GE healthcare beads (or the ones I know of), superdex offers the best 
resolution and mechanical strength (thus higher flow rate, very important for 
FPLC). GE healthcare’s competitors now also offer something similar to the 
superdex and prepacked superdex columns, so it’s worth doing a little research 
before the purchase. 

Compared to the superdex, sephadex is lower in resolution, and much lower in 
mechanical strength. The latter makes it not really a FPLC grade gelfiltration 
medium, but rather a gravity flow medium. It seems that sephadex nowadays are 
mainly used in desalting (spinning) columns, probably because in such 
situations the components to be separated have huge differences in size and the 
short spin columns do not complain about being compressed to 3/4 of its 
original length.

Between superdex and sepharose there are also superose, which is lower in 
resolution than superdex, but may allow you to separate something larger than 
600 kDalton, and Sephacryl, which I can not comment due to lack of experience 
with this product.


I think GE healthcare sells most of these beads in both bulk form (sold in 
little bottles) and prepacked column form, except for the top-resolution 
superdex beads (10 um beads or finer), which are sold only as prepacked columns 
(the 10 mm diameter columns and the 3.2 mm diameter columns). Packing a 
high-quality FPLC grade gel-filtration column does require some skill and 
equipment, so I recommend you to buy the prepacked columns. I like the 10 mm 
diameter ones because they are quite versatile. 16 mm and 26 mm columns allow 
you to load more sample each time, but they use larger beads hence are slightly 
lower in resolution, and for small quantity of sample, the dilution is 
excessive.

For the major characteristics of the GE (Pharmarcia) beads, please refer to the 
GE healthcare gelfiltration handbook linked below.  The chart on page 16 (Fig 
1.7. Gel filtration media fractionation range guide.) is very straight forward:

https://www.google.ca/url?sa=trct=jq=esrc=ssource=webcd=1ved=0CB0QFjAAurl=https%3A%2F%2Fwww.uni-ulm.de%2Fuploads%2Fmedia%2FGel_Filtration_02.pdfei=TdFWVYyROYj4yQTzg4GAAQusg=AFQjCNGNSSu1x9gWV8p7sXm7KoVdV_OJZQsig2=llZUhtkDytXo8QLNJqF_hwbvm=bv.93564037,d.b2wcad=rja

Sigma also sells some of these media (not limited to GE products), and they 
also have a clear, short chart for their characteristics:
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/life-science/proteomics-and-protein/proteomics/chromatography_gel_filtration.pdf


Zhijie




From: xaravich ivan 
Sent: Friday, May 15, 2015 11:51 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Superdex vs Sephadex

Hi fabulous cc4bbs, 
I had recently asked you about using HPLC or FPLC for protein purification.Now 
I have another question relating to that. I am trying to buy a new SEC column 
for the FPLC for protein purification but am getting some resistance from some 
experienced people in the lab who have now moved from promoting HPLC to 
sephadex beads instead of superdex column.

Could you please suggest what is the major difference between sephadex columns 
and superdex columns in your personal experience?  Are there any sephadex 
columns or just beads? I have always used superdex GE prepacked columns which 
have been fabulous for Gel filtration.

Please advise and as always thanks in advance.

ivan

Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!!!!!

[Zhijie Li]

The methods part of the 2008 nature paper also mentioned the two earlier, 
high-res structures 1F39 (1.9 A) and 1LMB (1.8 A), which together cover 
almost the whole strucutre except the linker regions.


I wonder, in such situations, is it a good practise to use the high res 
structures, either as referenece structures or as start points for 
refinement, to improve the low res structures? I have no doubt that most 
people would give both a try and compare the results. But what are people's 
opinions on publishing the resulting low res structures that contain 
significant amount of information coming from prior high-res strucutures? In 
which way should we think: this gives better structure so that the user 
sees less artifact or, no, this introduces information that did not come 
from my data?


Zhijie



-Original Message- 
From: Mark J van Raaij

Sent: Thursday, April 23, 2015 1:05 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!

The abstract of the papers says they used MIR.

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij








On 23 Apr 2015, at 18:57, Todd Jason Green wrote:

My guess is they had the best data they could get, did molecular 
replacement with the two halves of the repressor and the dna, got a 
solution and didn't use appropriate restraints in the refinement. Like 
Phoebe mentioned, we have better tools for this these days.




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Mark J van 
Raaij [mjvanra...@cnb.csic.es]

Sent: Thursday, April 23, 2015 11:49 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] 3BDN, 16.5% Ramachandran Outliers!

How reliable is too general a question - it depends on what you want to 
know.
At 3.9Å they could probably place the phosphate atoms quite well and see 
the general fold of the protein.
Finer details will be less reliable, i.e. where the exact side-chains are 
etc.
They could probably have forced more amino acids into favourable 
Ramachandran angles, but would that have made the structure better? 
Would these favourable angles have been more right? At 3.9Å you can't 
know for sure.
Would they have been able to draw more biological conclusions? I'd say 
not.
As long as they do not draw more conclusions in the paper than what is 
supported by the medium-resolution data, the structure provides useful 
information.


Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/~mjvanraaij








On 23 Apr 2015, at 18:03, Misbah ud Din Ahmad wrote:


Dear crystallographers,

The PDB entry
http://www.rcsb.org/pdb/explore.do?structureId=3BDN
has 16.5% Ramachandran outliers. When I opened this PDB file in coot and 
checked for Ramachandran outliers, the results are:

In preffered region: 58.04%
In allowed regions: 19.78%
Outliers: 22.17%  !

With an R-free of 37.4% at 3.9 A resolution, could you please tell me how 
reliable this structure of Lambda repressor bound to DNA is?



Thanks
Misbha

Re: [ccp4bb] how to recover my data

[Zhijie Li]

Smith,

As Ian said, mtzdump can give you some clues, but will not solve your problem. 

The file that mtzdump could not read apparently has changed length due to some 
sort of insertion. The other file that mtzdump could process has the correct 
length and the stats look reasonable, so it might be OK, although we still can 
not rule out the possibility that a few numbers might have been changed in the 
dataset. Have you tried to do anything with the latter file, such as a few 
rounds of refinement? What did you see?

If you send me the two mtz I can take a look and make some guesses. If the 
problem was as simple as introduction of some 0D 0A insertion/deletion it can 
be fixed. If you can consult the technician who recovered the data and tell us 
what software he used it would be helpful.

Zhijie



From: Smith Liu 
Sent: Saturday, March 07, 2015 9:24 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] how to recover my data

Thanks Ian. I got it from the bin folder you mentioned. A moment ago I have 
failed to localize it from the CCP4 graphical interface.

Smith






At 2015-03-07 21:13:58, Ian Tickle ianj...@gmail.com wrote:

  Hi Smith


  Not sure what you mean, the current version of CCP4 (as have all previous 
versions) certainly does contain mtzdump:


   which mtzdump
  /software/CCP4-6.5.0/ccp4-6.5/bin/mtzdump


  The limited output is intentional: mtzdump by default only lists the first 
10 reflexions, and you have exactly 10 in your list.  If you want more then 
supply an NREF value, e.g.


  echo nref 9 |mtzdump HKLIN my.mtz |less


  But mtzdump (or any MTZ utility for that matter) is not going to help you 
with a corrupted file, since the MTZ read routines assume that the file has a 
strictly defined format which has most likely been modified when it was 
transferred from the damaged disk, and is therefore now unreadable by any 
program that assumes the defined format.


  What does the technician who transferred the data have to say about it?  He 
is the only person who can possibly tell you how the file has been corrupted.  
What you need is a hex dump of the file (e.g. using 'hexdump') and someone who 
understands a) the correct format and b) exactly how it was corrupted.  Then it 
may be possible to write a bespoke program tor recover your data.  But that is 
not going to be easy: it will require someone with some expertise in 
programming.

  Attempting to read the file with random programs has even less chance of 
working than I have of winning the lottery!  As others have suggested there are 
much easier ways of recovering your data (such as reprocessing the images).


  Cheers


  -- Ian



  On 7 March 2015 at 06:29, Smith Liu smith_liu...@163.com wrote:

Dear All,

The current version of CCP4 does not contain mtzdump. I use UCLA MBI ― 
mtzdump to process it. For a mtz file, the output says there was no valid 
reflection data. For another mtz file, it only gave about 50 lines information 
(not a new mtz file). As for the original mtz is very large, I think at least 
some message in the mtz file has been missing or has not been recovered by UCLA 
MBI ― mtzdump.

Please feel free for further advise.

Smith


Follwing: output from the UCLAMBI-mtzdump



mtzdump - dump data from an MTZ reflection data file. A CCP4 program
Your mtzdump Results
Cell Dimensions : (obsolete - refer to dataset cell dimensions above)

  130.3260  130.3260  360.3400   90.   90.   90. 

 *  Resolution Range :

0.30.22676 (180.170 -  2.100 A )

 * Sort Order :

  1 2 3 0 0

 * Space group = 'I 4 2 2' (number 97)



  
1 ASC  0  62  0  100.00 32.5 32.5 180.17   2.10   H  H  
2 NONE 0  43  0  100.00 13.4 13.4 180.17   2.10   H  K  
3 NONE 0 171  0  100.00 64.4 64.4 180.17   2.10   H  L  
4 NONE0.019.0 0  100.00 9.51 9.51 180.17   2.10   I  
FreeR_flag  
5 NONE   65.8466807.5 47890   47.08 14985.84 14985.84  41.21   2.10   J  IMEAN  
6 NONE   22.7 37273.6 47890   47.08  1161.73  1161.73  41.21   2.10   Q  
SIGIMEAN  
7 NONE   65.8466807.5 50197   44.54 15601.05 15601.05  41.21   2.10   K  I(+)  
8 NONE   36.9 37273.6 50197   44.54  1723.38  1723.38  41.21   2.10   M  
SIGI(+)  
9 NONE   65.8466807.5 53778   40.58 16626.28 16626.28  41.21   2.10   K  I(-)  
10 NONE   36.9 37273.6 53778   40.58  1568.40  1568.40  41.21   2.10   M  
SIGI(-)  
11 NONE   67.3  6770.8 47890   47.08  1006.23  1006.23  41.21   2.10   F  F  
12 NONE5.6   446.6 47890   47.0876.6976.69  41.21   2.10   Q  SIGF  
13 NONE -514.8   537.4 56085   38.03 0.2187.57  41.21   2.10   D  DANO  
14 NONE0.0   380.1 56085   38.0391.6391.63  41.21   2.10   Q  
SIGDANO  
15 NONE   67.3  6770.8 50197   44.54  1032.56  1032.56  41.21   2.10   G  F(+)  
16 NONE8.2   446.6 50197   44.5490.1390.13  41.21   2.10   L  
SIGF(+)  
17 NONE   67.3  

Re: [ccp4bb] how to recover my data

[Zhijie Li]

Hi Ian,

Unix/Linux uses a single byte 0A (the linefeed character) as the line end 
marker for text, while DOS/Win use two bytes 0D 0A (carriage return + line 
feed). Old Mac systems use 0D. (what a mess!) 

So a simple UNIX to DOS operation should expand every 0A in a file to 0D 0A:
perl -pe 's/\n/\r\n/' input.mtz  output.mtz 

For MTZ file, since the main body of it is an array of REAL*4, apparently this 
operation will generate a lot of ‘insertional mutations’. No wonder the data is 
scrambled and the header cannot be found (its position is shifted, no longer at 
where the pointer points).

During the conversion from UNIX to DOS, there is a complication: there could be 
a few 0D 0A already in the original file. Smarter unix2dos programs will keep 
these 0D 0A unchanged so that there won’t be weird-looking 0D 0D 0A (for 
example http://www.thefreecountry.com/tofrodos/ states this in its source file 
and has this behavior). Some dumber methods will simply convert all of them 
into 0D 0D 0A, for example: 
perl -pe 's/\n/\r\n/' input.mtz  output.mtz


When converting from the DOS format back to UNIX format, in our binary file 
case, the dumber method will work but the smarter programs will cause problems. 
Because for all the 0D 0A in the DOS file generated by the smarter programs, 
there is no way to tell which used to be 0A and which used to be 0D 0A in the 
original, and they are all converted back to 0A. Therefore some 0D in the 
original file will be lost. With the dumber method, all 0D 0A were 0A in the 
original file, so there would be no problem changing them all back to 0A.


In a given MTZ, there will almost certainly be a lot of 0A. But 0D 0A could be 
rare or non-existent. So after a unix-dos then dos-unix conversion, the result 
depends on how many 0D 0A were there in the original file, and how the program 
did the UNIX-DOS conversion.

With things like the following, it should be OK:
perl -pe 's/\n/\r\n/' test.mtz  test1.mtz
perl -pe 's/\r\n/\n/' test1.mtz  test2.mtz

To test the dumber (perl) method, I used an MTZ file, which contains 7 0D 0A in 
the data section. Here is the result:
test.mtz 2030184 bytes: MTZdump OK
test1.mtz 2032486 bytes  : MTZdump error
test2.mtz 2030184 bytes  : MTZdump OK
cmp test.mtz test2.mtz : the two files are identical

With todos/fromdos (http://www.thefreecountry.com/tofrodos/ ):
test.mtz 2030184 bytes : MTZdump OK
todos.mtz 2032479 bytes : MTZdump error (note the size difference compared to 
test1.mtz, the 7 0D 0A in the original file were kept unchanged)
fromdos.mtz 2030177 bytes : MTZdump error (7 original 0D 0A were shrinked to 0A)


Ian, in your test with todos and tounix, it seems that the final MTZ still has 
a header information at the correct location, so that MTZdump could read it. 
But some of the numbers saved in the data array seem damaged, so some of the 
stats in MTZdump were out of range. It would be interesting to read the todos 
and tounix source code to see why that happened. Or with a binary file 
comparison tool we might be able to guess the cause by taking a look at the two 
files.

Zhijie






From: Ian Tickle 
Sent: Friday, March 06, 2015 5:59 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] how to recover my data

Hi, just for fun and to demonstrate what can go horribly wrong if you blindly 
use utilities that were specifically designed for changing the line terminators 
in an ASCII file, I applied these utilities to an MTZ file.


First I used 'todos' to simulate what I suspect the technician has done:


todos  original.mtz  original+todos.mtz


Then I used 'tounix' in an attempt to recover the original file, as others have 
suggested:

tounix  original+todos.mtz  original+todos+tounix.mtz


What can possibly go wrong?  The log files from mtzdump are attached - see for 
yourself! (the mtzdump on original+todos.mtz went into an infinite loop and I 
had to kill the process).


Cheers


-- Ian





On 6 March 2015 at 07:20, Smith Lee 
0459ef8548d5-dmarc-requ...@jiscmail.ac.uk wrote:

  Dear All,

  For the issue of the recovery of the mtz file, I have tried randomly to use 
excel to open one specific mtz file, however in this way all  the mtz files in 
the computer will have a excel icon (X), although the file extension is still 
.mtz. If I tried further to open one specific mtz file (with excel icon) with 
the notepad, all the mtz files will have the notepad icon. If I tried further 
to open one specific mtz file (with notepad icon) with the wordpad, all the mtz 
files will have the wordpad icon. 

  I hope these cluses can be helpful for you to give me the advise on recovery 
of mtz files.

  Smith



  On Thursday, March 5, 2015 11:51 PM, Robbie Joosten 
robbie_joos...@hotmail.com wrote:




  Hi Smith,

  If this is really the problem Ian describes, you can try the Linux programs 
unix2dos and dos2unix the change the line endings. A potential source of the 
problem might be copying the file with certain (S)FTP 

Re: [ccp4bb] how to recover my data

[Zhijie Li]

Hi Smith,

There is a viewHKL in ccp4, but that’s for viewing an intact MTZ. There are 
many tools for manipulating MTZ files in ccp4, but I doubt any of them was 
designed for you to fix a corrupt MTZ. 

You may try opening your MTZ with a Hex editor to see if its structure looks 
fine.
A Hex editor: http://www.flexhex.com/download/
(A very helpful feature of this software is that when you mouse-over a 
highlighted selection, it converts HEX numbers to DEC int or float.)


MTZ file is not very complicated. After a simple header saying “MTZ” and a 
number (multiply the int number at byte 5-8 by 4 you get the position of the 
header information), a marker and a few lines of 00, the main body of an MTZ is 
a simple data array with each data points occupying 4 bytes (a floating point 
number REAL*4). Then after this block of data, the “header information” (not 
the head of the file) are stored at the end of the file, starting with “VERS 
MTZ”.

In the data array block, since every line of data you see in view HKL starts 
with the H K L, you can easily locate the reflexion of interest by looking for 
the three simpler-looking numbers. 
For example: 
00 00 18 c2  is –38, 
00 00 00 00  is 0,
00 00 a0 40  is 5, 
So these are H K L –38 0 5 
You may also see a lot of ff fa 5a 5a in the data block. Those are unmeasured 
data I believe.

If you open your MTZ and cannot find the header information at the end then 
that means the file has lost a chunk. If you multiply the number stored at byte 
5-8 by 4 and at that address you do not see “VERS MTZ”, the files also has 
changed length, or has been partially overwritten after being deleted. 
For example, If you have 1B BC 07 00 at byte 5-8, that’s Hex 0007BC1B=506907, 
multiply by 4 you get 2027628=1EF06C. Go to address 1EF06C, the cursor should 
land on the M of “VERS MTZ”.

You can find descriptions of the MTZ format here:
http://www.ccp4.ac.uk/html/mtzformat.html
Zhijie


From: Smith Lee 
Sent: Thursday, March 05, 2015 3:46 AM
To: Zhijie Li 
Subject: Re: [ccp4bb] how to recover my data

Zhijie,

Is any mtz editor? I remember there is a software on it but I currently forget 
the name of that software.

Smith 



On Thursday, March 5, 2015 6:23 PM, Zhijie Li zhijie...@utoronto.ca wrote:




Hi Smith,

I wonder why after you copy and paste the content of PDB it became readable by 
Coot. It is possible that some sort of reading error was introduced to the PDB 
file during the recovery, which was then filtered out by the text editors 
(extended ASCII for example). What was the cause of the damage? Can you send me 
the PDB file that coot could not open? 

For the mtz files, it can be quite difficult to fix if there was a reading 
error during the recovery. Being binary certainly makes it difficult to be 
dealt with. If you still have the .sca files (if you used HKL2000) maybe 
starting from there is easier.

Zhijie



From: Smith Lee 
Sent: Thursday, March 05, 2015 12:36 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] how to recover my data


Dear All,

Recently my computer hardware has been broken and all the data has been 
recovered to movable hardware by technician. However I find the recovered PDB 
file and the MTZcould not be openned by Coot. Then I open the revovered PDB 
file by WordPad, and from WordPad I copied it to notepad and save it as pdb 
file. I find the Coot can open the notepad saved pdb file, thus my pdb files 
can be succesfully recovered from the hardware.

But will you please tell me how to have Coot open my mtz file? After data 
recovery by the technicial, the data size of the mtz file did not decrease, 
thus I think there is a way to have it recovered.

I have not noticed there were similar or identical posts as mine for recovery 
data before in the CCP4 mail list. 

Thus I am looking forward to getting a reply from you on how to recover my mtz 
file.

Smith

Re: [ccp4bb] Superposition of select residues

[Zhijie Li]

Hi Yanfeng,

You need to supply the two imol numbers in the apply_lsq_matches() function 
(defined in WinCoot\share\coot\python\coot_lsq.py).


For example:

clear_lsq_matches()
add_lsq_match(12,18,B,12,18,D,2)#match-type is 0 for all-atom, 1 
for mainchain and 2 for CAs

add_lsq_match(25,35,B,25,35,D,2)
apply_lsq_matches(0,1)#assuming imol 1 is 
the moving one and imol 0 is the reference


Zhijie

-Original Message- 
From: yanfeng zhou

Sent: Thursday, February 12, 2015 9:03 AM
To: Zhijie Li
Subject: Re: [ccp4bb] Superposition of select residues

Morning, Zhijie. Thanks for your tips.  They are really useful.

I have a quick question here about the mutli range LSQ in coot.  I
read the link you post, and how do I define the imol number in this
multi range lsq command?  It only listed chain id in the instruction.

Thanks,
Yanfeng 

Re: [ccp4bb] Superposition of select residues

[Zhijie Li]

Hi Oarabile,

Coot allows you to align structures by selected atoms:
https://www2.mrc-lmb.cam.ac.uk/Personal/pemsley/coot/docs/coot-faq.html#How-do-I-do-a-superposition-on-just-a-part-of-my-structure_003f
I guess what you are trying to do can be accomplished by the “multi-range LSQ 
superposition” method.

Note that the commands shown in the above page are in Guile/Scheme format, 
which is now not supported in Coot (if I am correct). You need to write the 
commands in python format like this:

superpose_with_atom_selection (0, //B/1-100”, 1, //B/1-100,0)
-basically: i)replace all ‘-’with ‘_’in function name, ii) put all parameters 
in a pair of brackets and iii) separate them with commas. 
For python formatting see 
https://www2.mrc-lmb.cam.ac.uk/Personal/pemsley/coot/web/docs/coot.html#Python

You can either make a little txt file “scripts.py” with these commands and let 
Coot run it, or you can type them one by one in Calculate-Scripting-python.

Zhijie

From: Kgosisejo, Oarabile 
Sent: Thursday, February 05, 2015 5:41 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Superposition of select residues

Hi all,

Does anyone know of a program to use to superpose only selected protein 
residues, e.g. enzyme active site residues. I solved my structure using 
molecular replacement now I want to compare its active site to those of 
homologous structures. I have used the DALI server before but I had to upload 
the whole model.

Thank you for your advise


Best Regards,


Oarabile M. Kgosisejo, MSc. Candidate
University of Saskatchewan

o.kgosis...@usask.ca

Re: [ccp4bb] chloride or water

[Zhijie Li]

Hi Jacob,
I would agree with Nat. I think with the 3A data and the local environment, 
putting a Cl there is only justified by the need of supressing a peak in the 
difference map. Yes the peak can really be a Cl or a water or a mixture of the 
two (I would like to do an experiment with my Cl containing data, which has Cl 
sites identified by iodide data, see what would happen when resolution is 
reduced and SNR is weakened). But we have to be aware that with 3A data there 
can be significant phase errors that lead to generation of features that are 
either not there or not so significant as it appears. In the past I have even 
seen a structure that puts a putative sugar (completely unknown type of 
glycosylation) in a blob in 3A structure because the authors thought the blob 
looked like the particular sugar residue. 
The practice of putting a ion or a small ligand simply for supressing 
difference peaks, to my view is not good practice for two reasons: 1) compared 
to protein residues, ions, and small ligands in low res cases, lack the 
geometric constraints and can be easily abused for the purpose of flattening 
the difference maps (admittedly a lot of water molecules is doing that, but we 
are told to be very careful with that already); 2) practically it makes 
searching/analysis of the structures in PDB a lot more difficult that it should 
be. It would be much easier for users to search an incomplete but reliable 
database than one that contains a mixture of real and wrong data, especially 
considering that not all users are experts on interpreting electron density 
maps or protein structures. 
Zhijie

From: Keller, Jacob 
Sent: Wednesday, January 21, 2015 1:17 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] chloride or water

I reiterate that assigning a chloride is not “wild speculation” or “just making 
something up” in light of what we know about the situation.

 

I see your point about not knowing that it’s a chloride, but I think you would 
agree that it is certainly more likely a chloride than map-noise, and perhaps 
more likely than water as well. Would you agree that chloride is the best 
guess, at least? What are the options for that blob, and what is the 
probability of each? I think you want to make sure people don’t get misled by 
it, which is a good point and a noble aspiration. I would argue that “not 
choosing” is here, as everywhere else, indeed choosing. And if you choose 
nothing here, you are almost certainly wrong, given the data. Some might be 
surprised or misled that a cavity like that would be totally empty, and the map 
density is unequivocal evidence that something is indeed there. So what now? 
Maybe a solution would be dummy atoms, maybe call them agnostons (agn) or 
something? Perhaps this is a basic disagreement about what a protein structure 
model represents, with one opinion being “that which we can rely upon 
confidently” and the other being “that which is most likely considering the 
data.” Each has advantages depending on one’s goals. Since the PDB is certainly 
tainted by structures modeled in accordance with the “most likely” outlook, one 
now has to be cautious about all structures.

 

I prefer the latter since I would try to be responsible/skeptical enough to go 
back to the original crystal data before making important conclusions based on 
it. Maybe there should be a disclaimer printed in each PDB file…

 

JPK

 

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols
Sent: Wednesday, January 21, 2015 12:33 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] chloride or water

 

On Wed, Jan 21, 2015 at 9:05 AM, Keller, Jacob kell...@janelia.hhmi.org wrote:

  Not sure why there is this level of suspicion about the poor halide when 
waters generally get assigned so haphazardly. I would say that there are 
probably more “wrong” waters in the PDB than wrong chlorides, but there’s not 
much fuss about that.

 

Great, so leave it empty instead of just making something up.  Perhaps future 
generations will figure out a more rigorous and quantitative method for 
handling such features than guessing based on screenshots posted to a mailing 
list.  At this resolution water placement is difficult to justify anyway - and 
since neither the scattering properties nor the coordination distances are 
especially accurate, trying to assign chemical identity in the absence of any 
supporting information (for example anomalous data) is especially futile.

(Although at least in this case the resolution is an obvious red flag - to a 
crystallographer, anyway - indicating that any lighter ions shouldn't be taken 
very seriously.  Other biologists, of course, may be more trusting.)

-Nat

Re: [ccp4bb] chloride or water

[Zhijie Li]

Quote:
(I would like to do an experiment with my Cl containing data, which has Cl 
sites identified by iodide data, see what would happen when resolution is 
reduced and SNR is weakened)

I just realized that this experiment has an inherent problem: artificially 
reducing the resolution cannot simulate the lack of order in low-diffracting 
crystals. Some large peaks in low-res structures in theory can arise simply 
because atoms in different ASU happened to have a high statistical appearance 
at that position, assuming that the phases are right. Correct me if I am wrong.

Zhijie



From: Zhijie Li 
Sent: Wednesday, January 21, 2015 2:12 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] chloride or water

Hi Jacob,
I would agree with Nat. I think with the 3A data and the local environment, 
putting a Cl there is only justified by the need of supressing a peak in the 
difference map. Yes the peak can really be a Cl or a water or a mixture of the 
two (I would like to do an experiment with my Cl containing data, which has Cl 
sites identified by iodide data, see what would happen when resolution is 
reduced and SNR is weakened). But we have to be aware that with 3A data there 
can be significant phase errors that lead to generation of features that are 
either not there or not so significant as it appears. In the past I have even 
seen a structure that puts a putative sugar (completely unknown type of 
glycosylation) in a blob in 3A structure because the authors thought the blob 
looked like the particular sugar residue. 
The practice of putting a ion or a small ligand simply for supressing 
difference peaks, to my view is not good practice for two reasons: 1) compared 
to protein residues, ions, and small ligands in low res cases, lack the 
geometric constraints and can be easily abused for the purpose of flattening 
the difference maps (admittedly a lot of water molecules is doing that, but we 
are told to be very careful with that already); 2) practically it makes 
searching/analysis of the structures in PDB a lot more difficult that it should 
be. It would be much easier for users to search an incomplete but reliable 
database than one that contains a mixture of real and wrong data, especially 
considering that not all users are experts on interpreting electron density 
maps or protein structures. 
Zhijie

From: Keller, Jacob 
Sent: Wednesday, January 21, 2015 1:17 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] chloride or water

I reiterate that assigning a chloride is not “wild speculation” or “just making 
something up” in light of what we know about the situation.

 

I see your point about not knowing that it’s a chloride, but I think you would 
agree that it is certainly more likely a chloride than map-noise, and perhaps 
more likely than water as well. Would you agree that chloride is the best 
guess, at least? What are the options for that blob, and what is the 
probability of each? I think you want to make sure people don’t get misled by 
it, which is a good point and a noble aspiration. I would argue that “not 
choosing” is here, as everywhere else, indeed choosing. And if you choose 
nothing here, you are almost certainly wrong, given the data. Some might be 
surprised or misled that a cavity like that would be totally empty, and the map 
density is unequivocal evidence that something is indeed there. So what now? 
Maybe a solution would be dummy atoms, maybe call them agnostons (agn) or 
something? Perhaps this is a basic disagreement about what a protein structure 
model represents, with one opinion being “that which we can rely upon 
confidently” and the other being “that which is most likely considering the 
data.” Each has advantages depending on one’s goals. Since the PDB is certainly 
tainted by structures modeled in accordance with the “most likely” outlook, one 
now has to be cautious about all structures.

 

I prefer the latter since I would try to be responsible/skeptical enough to go 
back to the original crystal data before making important conclusions based on 
it. Maybe there should be a disclaimer printed in each PDB file…

 

JPK

 

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Nat Echols
Sent: Wednesday, January 21, 2015 12:33 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] chloride or water

 

On Wed, Jan 21, 2015 at 9:05 AM, Keller, Jacob kell...@janelia.hhmi.org wrote:

  Not sure why there is this level of suspicion about the poor halide when 
waters generally get assigned so haphazardly. I would say that there are 
probably more “wrong” waters in the PDB than wrong chlorides, but there’s not 
much fuss about that.

 

Great, so leave it empty instead of just making something up.  Perhaps future 
generations will figure out a more rigorous and quantitative method for 
handling such features than guessing based on screenshots posted to a mailing 
list.  At this resolution water placement is difficult to justify anyway - and 
since

Re: [ccp4bb] A basic question about Fourier Transform

[Zhijie Li]

Hi Chen,

Here is what I think:

Assuming a crystal is perfect and is being shot at 0 K, then the maximum 
resolution one experiment can achieve is limited by the wavelength of the 
X-ray. It can’t be better than the half-wavelength under the normal 
experimental setting (minimum d=lambda/2/sin(90)=lambda/2). With shorter 
wavelength, we get more reflections (an Ewald sphere with larger radius 
encloses more lattice points), and that’s more sampling points and more 
information. With infinitely short wavelength, we get infinitely detailed 
information. On the other hand, the seemingly continuous, detailed profile we 
get from a single molecule diffraction is also limited (smeared) by the same 
X-ray wavelength. So it is only a difference between measuring many discrete 
points and measuring (smeared) continuity. 

In other words, the continuous curve we get from the single molecule 
diffraction experiment does not contain information with frequency higher than 
that of the X-ray. It only contains information with frequency up to that of 
the X-ray. Recalling that the minimum d from a crystal diffraction experiment 
is lambda/2, then for a 1-D crystal’s unit cell (with edge length a), we are 
sampling it with a frequency of 2a/lambda. I think the sampling theorem says 
that this sampling frequency is as good as the continuous curve we get from 
single molecule diffraction with X-ray of wavelength lambda.

With real life crystals, which are neither perfect, nor at 0 Kelvin, what makes 
difference is, by using a single molecule instead of a crystal, we can get away 
from the conformational differences of molecules found in a crystal, the 
defects in crystal, the heterogeneity of the crystal (e.g., the mosaicity), and 
probably even the background generated by solvent atoms (as the single molecule 
might be floating in vacuum). The packing defects and heterogeneity in a 
crystal is probably what limits resolution of our protein crystals in most 
cases. So when we are freed from the situation of having to use a crystal, in 
theory with short enough X-ray wavelength, by shooting at a single molecule 
that is not moving too much during the exposure, we can get a very high 
resolution that would not be achievable using a crystal. Now what weakens our 
high angle signal, limits the high resolution, and smears our map is the real 
thermo motion of the atoms, not the heterogeneity of the crystal. Then for the 
next step, with that highly sensitive detector and our ability of sending small 
pack of photons to the molecule, we might be able to get very quick snap shots 
of the molecule, essentially reducing the motion blur. Then in that case, what 
ultimately limits our resolution (or confidence of the measurement?) is 
probably the number of photons we can send to an atom before the absorbed 
energy significantly affect its location – may I say, a situation similar to 
that faced by the cryoEM people? 

Zhijie




From: Chen Zhao 
Sent: Tuesday, January 20, 2015 10:18 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] A basic question about Fourier Transform

Dear all,


I am sorry about this slightly off-topic question. I am now a graduate TA for 
crystallography course and one student asked me a question that I didn't ask 
myself before. I don't have enough knowledge to precisely answer this question, 
so I am seeking for help here.


The question is, as I rephrased it, assuming we are able to measure the 
diffraction pattern of a single molecule with acceptable accuracy and precision 
(comparable to what we have now for the common crystals), is it better than we 
measure the diffraction spots from a crystal, given that the spots are just a 
sampling of the continuous pattern from a single molecule and there is loss of 
information in the space between the spots that are not sampled by the lattice? 
Of course this is more of a thought experiment, so we don't need to consider 
that all measurement is discrete in nature owing to the limitation of the pixel 
size. I kinda agree with him and I have a feeling that this is related to the 
sampling theorem. I do appreciate your valuable comments. If this is not true, 
why? If this is true, what is its effect on electron density?

Thank you so much for your attention and your help in advance!

Best,
Chen

Re: [ccp4bb] chloride or water

[Zhijie Li]

Hi Rohit,

If it is of some significance you may consider replacing NaCl with KI or NaI in 
your crystallization condition and collect a dataset at 1.5-2 Angstrom. The 
Iodide anomalous signal might be of some help for identifying Chloride binding 
sites. 
Without further evidence for a Cl ion, unless the environment strongly suggests 
an anion binding site (as what Robbie said) my personal view is that it is more 
proper to put a water there. 

Zhijie



From: rohit kumar 
Sent: Tuesday, January 20, 2015 1:59 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] chloride or water


Dear all,

I am solving a data of 3.0 Å resolution. In the active site we found an 
unidentified blob. The crystallization condition is (PEG 3350-25%, 
Naformate-200mM, MES (100mM)-6.5pH, and Nacl-200mM). 
Can anyone suggest what it could be? If I suppose that, it is chloride, how 
could someone differentiate between a chloride moiety and water.
below I am showing the coot figure in two different orientations.

Thanks in advance...



 




 





-- 

WITH REGARDS
Rohit Kumar Singh
Lab. no. 430,
P.I. Dr. S. Gourinath,
School of Life Sciences,
Jawaharlal Nehru University
New Delhi -110067

Re: [ccp4bb] Adding a residue with an insertion code

[Zhijie Li]

Hi Jared and all,

There is also a JAVA program called PDBeditor by Jonas Lee that may make the 
PDB editing part a little easier or less error prone: 
http://sourceforge.net/projects/pdbeditorjl/
I did a little test and find it pretty useful, especially for those who do not 
have access to a text editor that supports column editing mode (or those who 
haven’t yet get acquainted with notepad++). It allows you to select certain 
blocks in the PDB based on certain criteria, and edit properties of certain 
residues together, e.g., adding “A” to column 27 of residue 101. 

HTH

Zhijie


From: Sampson, Jared 
Sent: Wednesday, January 14, 2015 3:48 PM
To: Zhijie Li 
Cc: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Adding a residue with an insertion code

Thanks to Paul and Zhijie for the suggestions.   

In the past, I’ve also renumbered everything from 1 (using pdbset) for the 
duration of refinement, then renumbered the structure at the end.  For that, I 
would use either pdbset and a text editor (which is somewhat tedious, as there 
are several insertion positions in either the Kabat or Chothia numbering 
schemes, although Matt Franklin suggested to me off-list that their usage in 
new PDB deposits has been declining), or if I’m not feeling particularly 
secretive about the structure, via Andrew Martin's Abnum server 
(http://www.bioinf.org.uk/abs/abnum/), although that’s only good for antibody 
structures, of course.

In any case, insertion codes are the exception to the rule, and I don’t 
particularly mind the workarounds every now and then.  I just wanted to make 
sure I wasn’t missing an easier way to do it.  :)

Thanks again,

Jared
--
Jared Sampson
Xiangpeng Kong Lab
NYU Langone Medical Center
http://kong.med.nyu.edu/







On Jan 14, 2015, at 12:52 PM, Zhijie Li zhijie...@utoronto.ca wrote:


  Hi Jared,

  The only thing that comes to my mind that might help a little is to use 
pdbset to renumber your residues. Then you only need to change residue 101 to 
100A manually in a text editor (unfortunately I could not find a program that 
does that). 
  Here are the steps I would take:
  1) build the model with normal numbering, i.e., the concerned region would be 
100,101,102;
  2) manually change 101 to 100A in text editor;
  3) pdbset xyzin old.pdb xyzout new.pdb
  renumber increment –1 102 to 500 chain A  !move 
everything up starting from 102
  end

  You can make a little shell script file for step 3, to save some typing/typo. 
(see attachment for an example)

  Zhijie

  From: Sampson, Jared 
  Sent: Wednesday, January 14, 2015 11:04 AM
  To: CCP4BB@JISCMAIL.AC.UK 
  Subject: [ccp4bb] Adding a residue with an insertion code

  Along the same lines as Dialing’s recent post about inserting a residue, I’d 
like to ask: Is it possible to insert a residue with an insertion code in the 
residue number?  For example, what’s the best way to insert residue 100A 
between residues 100 and 101? 

  This situation arises frequently for me in antibody CDR loops, for example.  
I ran into it most recently just last week, and Coot wouldn’t allow this kind 
of insertion, even though the consecutively numbered residues were too far 
apart to be covalently linked.  (In the terminal window, it explained that 
residue was not at a terminus.”)  My workaround has been either to increment 
the residue numbers of the C-terminal portion of the chain, then renumber 
manually in a text editor; or, to position a lone amino acid in approximately 
the right spot, save the coordinates, copy them manually into the working PDB 
and adjust the numbering in a text editor, reload the PDB in Coot, and real 
space refine the loop with the insertion.

  Any suggestions for how to do this more efficiently?  Thanks!

  Cheers,
  Jared

  --
  Jared Sampson
  Xiangpeng Kong Lab
  NYU Langone Medical Center
  http://kong.med.nyu.edu/







  On Jan 14, 2015, at 9:54 AM, Emilia C. Arturo (Emily) ec...@drexel.edu 
wrote:


If the residues are consecutively numbered (Calculate  Renumber residues), 
and are assigned the same chain ID (Calculate  Change Chain ID), Coot might 
surprise you and link them on its own.

On Wed, Jan 14, 2015 at 9:39 AM, Zhijie Li zhijie...@utoronto.ca wrote:

  Have you done it?
  1) Click “add residue...”
  2) Click that “real space refine zone” button. 
  You will see what will happen.


  From: Dialing Pretty 
  Sent: Wednesday, January 14, 2015 9:24 AM
  To: CCP4BB@JISCMAIL.AC.UK 
  Subject: [ccp4bb] Coot: How to connect N-terminal to neighbouring 
C-terminal

  Dear All,

  Suppose I delete a residue ( residue 100 for example) for outlier 
refinement, then I add the same residue at the C-terminal of residue 99 (by Add 
terminal residue function of the Coot). By coot, how can I connect the 
C-terminal of residue 100 to the N-terminal of residue 101?

  I am looking forward to getting your reply.

  DIaling


  renumber.sh

Re: [ccp4bb] Coot: How to connect N-terminal to neighbouring C-terminal

[Zhijie Li]

Have you done it?
1) Click “add residue...”
2) Click that “real space refine zone” button. 
You will see what will happen.


From: Dialing Pretty 
Sent: Wednesday, January 14, 2015 9:24 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Coot: How to connect N-terminal to neighbouring C-terminal

Dear All,

Suppose I delete a residue ( residue 100 for example) for outlier refinement, 
then I add the same residue at the C-terminal of residue 99 (by Add terminal 
residue function of the Coot). By coot, how can I connect the C-terminal of 
residue 100 to the N-terminal of residue 101?

I am looking forward to getting your reply.

DIaling

Re: [ccp4bb] Adding a residue with an insertion code

[Zhijie Li]

Hi Jared,

The only thing that comes to my mind that might help a little is to use pdbset 
to renumber your residues. Then you only need to change residue 101 to 100A 
manually in a text editor (unfortunately I could not find a program that does 
that). 
Here are the steps I would take:
1) build the model with normal numbering, i.e., the concerned region would be 
100,101,102;
2) manually change 101 to 100A in text editor;
3) pdbset xyzin old.pdb xyzout new.pdb
renumber increment –1 102 to 500 chain A  !move 
everything up starting from 102
end

You can make a little shell script file for step 3, to save some typing/typo. 
(see attachment for an example)

Zhijie

From: Sampson, Jared 
Sent: Wednesday, January 14, 2015 11:04 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Adding a residue with an insertion code

Along the same lines as Dialing’s recent post about inserting a residue, I’d 
like to ask: Is it possible to insert a residue with an insertion code in the 
residue number?  For example, what’s the best way to insert residue 100A 
between residues 100 and 101? 

This situation arises frequently for me in antibody CDR loops, for example.  I 
ran into it most recently just last week, and Coot wouldn’t allow this kind of 
insertion, even though the consecutively numbered residues were too far apart 
to be covalently linked.  (In the terminal window, it explained that residue 
was not at a terminus.”)  My workaround has been either to increment the 
residue numbers of the C-terminal portion of the chain, then renumber manually 
in a text editor; or, to position a lone amino acid in approximately the right 
spot, save the coordinates, copy them manually into the working PDB and adjust 
the numbering in a text editor, reload the PDB in Coot, and real space refine 
the loop with the insertion.

Any suggestions for how to do this more efficiently?  Thanks!

Cheers,
Jared

--
Jared Sampson
Xiangpeng Kong Lab
NYU Langone Medical Center
http://kong.med.nyu.edu/







On Jan 14, 2015, at 9:54 AM, Emilia C. Arturo (Emily) ec...@drexel.edu wrote:


  If the residues are consecutively numbered (Calculate  Renumber residues), 
and are assigned the same chain ID (Calculate  Change Chain ID), Coot might 
surprise you and link them on its own.

  On Wed, Jan 14, 2015 at 9:39 AM, Zhijie Li zhijie...@utoronto.ca wrote:

Have you done it?
1) Click “add residue...”
2) Click that “real space refine zone” button. 
You will see what will happen.


From: Dialing Pretty 
Sent: Wednesday, January 14, 2015 9:24 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Coot: How to connect N-terminal to neighbouring C-terminal

Dear All,

Suppose I delete a residue ( residue 100 for example) for outlier 
refinement, then I add the same residue at the C-terminal of residue 99 (by Add 
terminal residue function of the Coot). By coot, how can I connect the 
C-terminal of residue 100 to the N-terminal of residue 101?

I am looking forward to getting your reply.

DIaling



renumber.sh
Description: Binary data

Re: [ccp4bb] off-topic;Crystal cannot dissolved in buffer

[Zhijie Li]

Hi Xiao,

I would poke the crystal with a piece of glass fiber or an oocyte needle to 
make sure it is not salt. Some protein crystals can be a little tough 
(rubber-like), but they react to poking very differently from rocks (salts) and 
will eventually shatter. So far I have found this test to be the most reliable 
before an X-ray diffraction image. You can also try IsIt (0.1% methylene blue) 
staining, glutaraldehyde crosslinking/staining (causing protein crystals to 
become yellow due to Schiff base formation) and so on. 
If you can see a band on SDS-PAGE gel and still want to confirm that it is your 
protein, you may consider MALDI-MS which is very sensitive, maybe too sensitive 
(bear in mind that due to potentially insufficient wash, the protein band or 
MALDI peaks may come from the solution not the crystal).

Nevertheless, shoot the crystal!

Zhijie



From: Xiao Xiao 
Sent: Thursday, September 25, 2014 7:53 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] off-topic;Crystal cannot dissolved in buffer

Hi everyone, 

Sorry for an off-topic question.

I got a problem with crystal dissolving. Basically I got crystals of my protein 
in various conditions, most conditions contain PEGs but different salts. These 
crystals has very similar shape, so it should not be salt.

Now I am trying to dissolve the crystal to make sure it is my protein, by 
SDS-PAGE and N-term sequencing. I washed the crystals in its original 
crystallization buffer few times then transfered them into regular buffer 
(500mM NaCl, 50mM HEPES 7.5) with or without 10mM DTT, however the crystal 
didn't dissolve.

I then tried to heat it then add SDS loading buffer to run a gel, I did see 
very small amount of protein on the gel, at the correct position, but it's not 
enough for N-term sequencing.

Is it normal for a protein crystal? And does anyone have any suggestion for 
dissolving such crystals?

Re: [ccp4bb] [phenixbb] local BLAST server

[Zhijie Li]

Hi Rob,

The BLAST nr database (fasta format) can be downloaded from the NCBI ftp:
ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/
As I remember it is the nr.gz file. When unzipped the file is called nr.

According to BLAST the nr database does contain PDB entries.
http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=WebPAGE_TYPE=BlastDocsDOC_TYPE=ProgSelectionGuide

It is significantly larger than the PDB data file you are currently using. 
You might consider extract all the PDB sequences from it so that you do not 
need to go through all the non-PDB sequences.


Zhijie



-Original Message- 
From: R.D. Oeffner

Sent: Friday, August 08, 2014 10:14 AM
To: pheni...@phenix-online.org
Subject: [phenixbb] local BLAST server

Hi,

I'm in the process of installing a local BLAST server for doing blast
protein queries. As I understand it I need a file with all the FASTA
sequences as input for initially generating my local BLAST database.
The one present in
ftp://ftp.rcsb.org/pub/pdb/derived_data/pdb_seqres.txt seems to
contain redundant entries. Querying it produces many extra PDB
chain-ids when compared to a BLAST query on the NCBI web server.

Does anyone know where to get a non-redundant version of FASTA records
so that I can create a similar database as the one used by NCBI?


Many thanks,

Rob

--
Robert Oeffner, Ph.D.
Research Associate, The Read Group
Department of Haematology,
Cambridge Institute for Medical Research
University of Cambridge
Cambridge Biomedical Campus
Wellcome Trust/MRC Building
Hills Road
Cambridge CB2 0XY

www.cimr.cam.ac.uk/investigators/read/index.html
tel: +44(0)1223 763234
mobile: +44(0)7712 887162
___
phenixbb mailing list
pheni...@phenix-online.org
http://phenix-online.org/mailman/listinfo/phenixbb 

Re: [ccp4bb] [phenixbb] local BLAST server

[Zhijie Li]

Hello,

My apologies for sending the previous post to the wrong BB.

Zhijie


-Original Message- 
From: Zhijie Li

Sent: Friday, August 08, 2014 11:10 AM
To: R.D. Oeffner ; CCP4BB@JISCMAIL.AC.UK
Subject: Re: [phenixbb] local BLAST server

Hi Rob,

The BLAST nr database (fasta format) can be downloaded from the NCBI ftp:
ftp://ftp.ncbi.nlm.nih.gov/blast/db/FASTA/
As I remember it is the nr.gz file. When unzipped the file is called nr.

According to BLAST the nr database does contain PDB entries.
http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=WebPAGE_TYPE=BlastDocsDOC_TYPE=ProgSelectionGuide

It is significantly larger than the PDB data file you are currently using.
You might consider extract all the PDB sequences from it so that you do not
need to go through all the non-PDB sequences.

Zhijie



-Original Message- 
From: R.D. Oeffner

Sent: Friday, August 08, 2014 10:14 AM
To: pheni...@phenix-online.org
Subject: [phenixbb] local BLAST server

Hi,

I'm in the process of installing a local BLAST server for doing blast
protein queries. As I understand it I need a file with all the FASTA
sequences as input for initially generating my local BLAST database.
The one present in
ftp://ftp.rcsb.org/pub/pdb/derived_data/pdb_seqres.txt seems to
contain redundant entries. Querying it produces many extra PDB
chain-ids when compared to a BLAST query on the NCBI web server.

Does anyone know where to get a non-redundant version of FASTA records
so that I can create a similar database as the one used by NCBI?


Many thanks,

Rob

--
Robert Oeffner, Ph.D.
Research Associate, The Read Group
Department of Haematology,
Cambridge Institute for Medical Research
University of Cambridge
Cambridge Biomedical Campus
Wellcome Trust/MRC Building
Hills Road
Cambridge CB2 0XY

www.cimr.cam.ac.uk/investigators/read/index.html
tel: +44(0)1223 763234
mobile: +44(0)7712 887162
___
phenixbb mailing list
pheni...@phenix-online.org
http://phenix-online.org/mailman/listinfo/phenixbb 

Re: [ccp4bb] protocol for seleno-methionine incorporation...

[Zhijie Li]

Hi Mintu,

I suggest using purified whole protein for ESI-MS. You will get a series of 
protein peaks (if the substitution is not 100%, which is often the case), each 
differ for 47 Dalton. You can determine how many Se each peak corresponds to if 
you know the molecular weight of your protein. Then you can integrate the peaks 
to get an overall incorporation percentage. 

Zhijie


From: Mintu Chandra 
Sent: Monday, June 23, 2014 11:57 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] protocol for seleno-methionine incorporation...

Dear all, 
Is there any standard protocol to know the number of incorporated 
seleno-methionine in a protein ??? I am growing one of the protein in minimal 
media for phase determination. 
I am using mass spectrometry facility to know the incorporated 
seleno-methionine but I wanted to know whether I should use the purified 
protein  or  digested protein for mass spectrometry.

Thanks,

Mintu Chandra.


-- 
Mintu Chandra
Senior Research Fellow
IISER BHOPAL
mi...@iiserb.ac.in
+918085288853 

Re: [ccp4bb] HisTrap Trap

[Zhijie Li]

Hi Bernhard,

I too suspect that it is some kind of metal chelating reagent causing the 
problem (possibly used in medium for carrying Fe2+, as the free Fe2+ is toxic 
to cells). One simple test would be to load a liter of the unused medium to the 
Ni2+ column and see what happens. Do you concentrate your medium before 
pull-down? If it is the chelating reagent in the medium, then concentrating the 
medium 10-30x may help a lot (also helps yield, since every affinity binding 
has a Kd). We do that regularly on tangential flow filter columns. 

It is a little weird that your first run was OK but the later ones suffered 
from Ni2+ loss. I wonder if you can try stripping the column with EDTA first 
and then loading it again with fresh NiCl2, every time before loading the 
medium. The reason to strip it is that I also worry that some Ni2+ on your 
column might have been partially replaced by some metal ions from the medium. 
(Loading 1L medium to a 1mL column does not sound like a great idea to me 
anyways...)


Zhijie



From: Bernhard Rupp 
Sent: Monday, May 19, 2014 10:13 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] HisTrap Trap

Hi Fellows,

 

my lab mates successfully expressed a glycoprotein in CHO cells in serum free 
medium, and

the protein captures nicely on HisTrap Excel 1ml columns (obviously, high yield 
is not my problem…).

We load ca 1L supernatant at 0.5 ml/min, and eluate with a steep imidazole 
gradient. 20mM Imidazole buffer for regeneration.

Works fine (and often…see yield remark).

 

Overcome by common crystallographers’ greed (nor creed), we switched to stable 
xfected HEK293, and cell free medium Gibco CD 293.

The first run gave high final yields  cheers. 

The second run less of either, because the small HisTrap column essentially 
dissolved – the medium collapsed, 

Ni leaches out, kaput as kaput goes. 

A 3rd run on a similar previously working column lead to the same result.

 

Only thing changed was the cells and medium. Same buffers, same gradients, same 
Akta equipment, same lab techs.

 

Before I improve the statistics by ruining further columns, has anybody 
experienced such a calamity that might

be blamable on secret media components or similar? There is a mysterious 
‘proprietary dispersant’ preventing

cell adhesion quoted….

 

Best wishes, BR

 



Bernhard Rupp 

b...@ruppweb.org

b...@hofkristallamt.org

http://www.ruppweb.org/

---

 

 

Re: [ccp4bb] Stereo monitors for use with Pymol and Coot

[Zhijie Li]

I beg to differ on this:
Also, passive screens have a pol-filter in place, the fine lines of which you 
will observe on a white background, the more disturbing the closer the viewing 
distance to the screen is. So, for general office applications (writing text), 
the screens are less useful. 
Our LG D2342P has no issue with office work. I never noticed any thin lines on 
the screen. In fact I think its 2D display is excellent. I wonder if the issue 
you have is related to the screen size: the pixels of 27 in screens are bigger 
than our 22 in screens.
Zhijie
From: mesters 
Sent: Thursday, March 06, 2014 3:55 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Stereo monitors for use with Pymol and Coot

Hi,

this is probaly due to the transition from old TFT style to new IPS panel based 
monitors... Several new passive 3d monitors are hitting the market such as AOC 
d2769Vh and the Philips Gioco 278G4. Both are based on 27 IPS Monitor panels. 

A list of possible monitors can be found at Tridef (many are old but new models 
are listed),  https://www.tridef.com/products/pc-licensed-products.

Problem with passive stereo is, you will half the resolution in the vertical 
direction. It is a problem if you are looking at wire-models of structures in 
pymol and especially fine-wire electron density mesh and models in coot as 
those noticably loose resolution compared to active stereo screens. Also, 
passive screens have a pol-filter in place, the fine lines of which you will 
observe on a white background, the more disturbing the closer the viewing 
distance to the screen is. So, for general office applications (writing text), 
the screens are less useful. This is not to big a problem for viewing full 
screen pictures, games and movies (increased distance to the screen...).

Moreover, with passive monitors, as the stereo effect increases with the screen 
size, the picture looks more pixeled compared to active stereo screens. I 
personally own a AOC d2769Vh and for 3D movies it is great, for coot not that 
useful if you plan longer sessions. At work, we operate an ASUS VG278HR (active 
stereo and build in emitter for glasses). Many hardware testers consider this 
screen the best one available on the market.

If you mainly need it for coot, I recommend to change your priorities and buy 
an active stereo screen such as Asus VG248QE or Asus VG278HR.
You do not need an expensive quadro card (600 will do fine) as the VG278HR has 
build-in emitter for operation with cheap nvidia glasses.
It pays off in the long run to invest a few more dollars as you (I assume) will 
spend a lot of time in front of the this device (so buy the best as you only 
have one pair of eyes).

- J. -




Am 05.03.14 23:46, schrieb Shaun Lott:

A rather US-centric question on passive 3D monitors...

I'm just getting set up in the US, and I'm surprised on how few passive 3D 
monitors seem to be around - many models seem listed as 'out of stock' when 
looking in the usual places (Amazon, NewEgg, BestBuys, Walmart etc.)

The best deal I have found is for an LG D2343PB-BN 
(http://www.lg.com/us/commercial/lcd-computer-monitors/lg-D2343PB-BN) at US$274

Does anyone have any experience with this model, or any suggestion about where 
best to buy 3D monitors in the US?

many thanks in advance

Shaun



-- 
Dr. Jeroen R. Mesters
Deputy, Senior Researcher  Lecturer

Institute of Biochemistry, University of Lübeck
Ratzeburger Allee 160, 23538 Lübeck, Germany

phone: +49-451-5004065 (secretariate 5004061)
fax: +49-451-5004068

http://www.biochem.uni-luebeck.de
http://www.iobcr.org

  
--
If you can look into the seeds of time and tell which grain will grow and which 
will not, speak then to me who neither beg nor fear (Shakespeare's Macbeth, Act 
I, Scene 3)
-- 
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Re: [ccp4bb] Aggregation assay

[Zhijie Li]

Hi,

Two things i would like to add:

1) Due to the dependence of A280 on amino acid composition, a simple 
two-wavelength 280 and 340 or 320 comparison is not very ideal for 
determining the scatter component of the UV absorption of protein/protein 
aggregate particles (the usefulness of this simple method for determining 
the degree of aggregation is not questioned). Instead, we use data points in 
the 320-350nm range from a UV absorption/scattering scan to plot a curve for 
determining the scattering component, which serves two purposes: a) to be 
used for subtracting the A280 to get real UV absorption of the protein at 
280nm, b) to have an idea of how aggregated the sample is.


To do this, we plot a double log curve with absorption values A and 
wavelengths lambda at 320 330 340 350 nm, and fit it to the equation

log(A)=C - k*log(lambda)
Then the resulting equation can be used for extrapolating the scatter 
component at 280 nm. Of course the larger this component is, the more 
aggregated the protein is. The degree of aggregation (particle size and 
population) is also reflected by the k value. The larger the k is, the less 
aggregated the solution is.


An explanation of the theory behind this can be found here:
http://www.chem.agilent.com/Library/applications/59633927.pdf
Our favorite method is the one in the scattering model part, note that in 
figure 7 their molecule has UV absorption peak at 300, while in most of our 
cases, the proteins have peak at 280. There should be an academic article on 
this method. However I can not locate it right now. Apologies for that.


In fact, a quick look at the UV curve in the 300-350 nm range can directly 
tell us how badly aggregated a protein solution is. A perfectly not 
aggregating protein solution should have a UV absorption curve that quickly 
drops to nearly zero at 300 nm. One can test this by measuring an 
aggregation sample before and after filtration or centrifugation and see the 
dramatic change on the UV curve.  (0.22um filter's pore sizes are quite 
comparable to the 200-400nm UV wavelengths, while most globular proteins are 
roughly 2-10 nm in diameter.)



2) Nanodrop's baseline function in the scan mode (Protein 280 for example) 
is actually an all-point zeroing. At the blanking step, the instrument 
records a blank absorption curve and later uses this curve to subtract the 
sample curve. So when using a proper solution as blank, the sample curve is 
quite faithfully reflecting the UV absorption + scattering. I believe most 
UV spectrometers allow the users to do the same in the scan mode. So they 
are all good for this task.


Zhijie



-Original Message- 
From: Michael C. Wiener

Sent: Friday, February 21, 2014 12:16 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Aggregation assay

Analogous to Dr. Mirella, we've used the ratio of A320/A280 (for membrane 
proteins). In response to a fussy reviewer, I located some relevant 
references (refs. 10-13, copied below) when we referred to this in A 
high-throughput differential filtration assay to screen and select 
detergents

for membrane proteins, Vergis et al., Anal. Biochem 407:1 (2010).

[10] S.J. Leach, H.A. Scheraga, Effect of light scattering on ultraviolet 
difference

spectra, J. Am. Chem. Soc. 82 (1960) 4790–4792.
[11] Y. Cordeiro, F. Machado, L. Juliano, M.A. Juliano, R.R. Brentani, D. 
Foguel, J.L.

Silva, DNA converts cellular prion protein into the b-sheet conformation and
inhibits prion peptide aggregation, J. Biol. Chem. 276 (2001) 49400–49409.
[12] G.J. Lee, A.M. Roseman, H.R. Saibil, E. Vierling, A small heat shock 
protein stably

binds heat-denatured model substrates and can maintain a substrate in a
folding-competent state, EMBO J. 16 (1997) 659–671.
[13] Y. Panyukov, I. Yudin, V. Drachev, E. Dobrov, B. Kurganov, The study of
amorphous aggregation of tobacco mosaic virus coat protein by dynamic light
scattering, Biophys. Chem. 127 (2007) 9–18.

Regards,

-MW

Michael C. Wiener, Ph.D.
Professor
Department of Molecular Physiology
and Biological Physics
University of Virginia
PO Box 800886
Charlottesville, VA 22908-0886
434-243-2731
434-982-1616 (FAX)

On Fri, 21 Feb 2014 15:05:46 +
Tanner, John J. tanne...@missouri.edu wrote:
I am aware of an assay for aggregation (aggregation rate) that is based on 
fluorescence measurements.  It involves excitation at 280 and emission in 
the range 260-400. Is there a reference for the absorbance method?


See

Nominé Y, Ristriani T, Laurent C, Lefèvre JF, Weiss E, Travé G. A 
strategy for optimizing the monodispersity of fusion proteins: application 
to purification of recombinant HPV E6 oncoprotein. Protein Eng. 2001 
Apr;14(4):297-305. PubMed PMID: 11391022.


http://www.ncbi.nlm.nih.gov/pubmed/11391022

Jack Tanner

Sent from Jack's iPad

On Feb 21, 2014, at 7:23 AM, Vivoli, Mirella 
m.viv...@exeter.ac.ukmailto:m.viv...@exeter.ac.uk wrote:


Dear Prerana,

before starting the crystallization you could try to check the 

Re: [ccp4bb] Recovering crystals from dry drops

[Zhijie Li]

Hi Debasish,

I would first use some of those crystals to make seeds and grow some new 
crystals so that I would not lose the crystal. Dehydration, even done 
systematically (eg, 
http://www.mitegen.com/mic_catalog.php?c=jenCrystaloptdehydrate), may or may 
not improve the diffraction. Like most other things in biological 
crystallography, it varies from case to case. I do not think other people’s 
experience really means anything for your crystals.

Zhijie


From: Debasish Chattopadhyay 
Sent: Thursday, February 20, 2014 10:59 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Recovering crystals from dry drops

Would you please share your experience and comments on recovering protein 
crystals from dry (or almost dry hanging drops) for data collection.

 

I found some beautiful crystals in hanging drops that were set up three years 
ago; from the color of the crystals ( the protein binds a colored substrate) 
and the their shape I know these are crystals of my protein.  I had collected 
data using some of these crystals when they were fresh; resolution was poor and 
the overall I/sigma was low.  I am curious if the dehydration would improve the 
diffraction now.

 

Thanks

 

Debasish

 

 

 

Ph: (205)934-0124; Fax: (205)934-0480

 

Re: [ccp4bb] Can not see density map when I turn off normalization in PYMOL

[Zhijie Li]

Hi Hongshi,

I think Dale is right. When you turn off the normalization, the level value you 
need to put in the isomesh command should be the absolute electron density 
value of the map instead of how many RMSD.

In your command window log, this line shows the range of the density values in 
your map:
ObjectMap: Map read. Range: -0.511 to 0.616
As you can see the highest value is 0.616, no wonder you see nothing when you 
tell the program to cut off at 3.0.

And the line above it shows the RMSD, marked as stdev:
ObjectMapCCP4: Current mean = -0.66 and stdev = 0.074981.

So for RMSD=3.0, the density cut off of this map is 0.075x3.0=0.225. Your 
command then is: isomesh fo-fc_ligand, bdligand002, 0.225
Or you can look at the coot window to find out what the density cut off is 
being used (map level=x. e/A^3 ), if you are not too sure about the RMSD 
calculation. 

An additional note: the same applies to UCSF Chimera, where the volume data 
(maps) control also requires you to specify the cut off or gradient in real map 
values instead of RMSD.

Zhijie


From: hongshi WANG 
Sent: Wednesday, February 19, 2014 3:47 PM
To: Zhijie Li 
Subject: Re: [ccp4bb] Can not see density map when I turn off normalization in 
PYMOL

Hi Zhijie, 

Thanks for your help.
Once i turned off the normalization in pymol. I could not see any density map. 

hongshi



On Wed, Feb 19, 2014 at 11:21 AM, Zhijie Li zhijie...@utoronto.ca wrote:

  Hi Hongshi,

  If you just put 
  isomesh fo-fc_ligand, omitmap, 3
  do you seem anything in the unit cell?

  If you do then please check if around your ligand there is any density.

  Zhijie



  From: hongshi WANG 
  Sent: Wednesday, February 19, 2014 12:30 PM
  To: CCP4BB@JISCMAIL.AC.UK 
  Subject: [ccp4bb] Can not see density map when I turn off normalization in 
PYMOL

  Hello there,



  I am making a fo-fc map for one ligand using pymol. I strictly followed the 
pymol wiki protocol (Display CCP4 Maps). Finally, I can get the ligand map 
using command:

  isomesh fo-fc_ligand, omitmap, 3, ligand, carve=2. 

  However, the problem is the map I got from pymol is smaller than the one I 
can see in coot at the same contour level (3.0).  

  So I gave a second trial based on the assumption that it may be caused by the 
mis-normalization.  I input the command: “unset normalize_ccp4_maps” to stop 
PyMOL from normalizing a cpp4 map. After that I loaded my ccp4 map file and 
tried to do the same things as what I did for the first time. But I could not 
see any mesh net (density map) shown up. I check the command window.

  PyMOLunset normalize_ccp4_maps

  Setting: normalize_ccp4_maps set to off.

  ObjectMapCCP4: Map Size 134 x 128 x 122

  ObjectMapCCP4: Map will not be normalized.

  ObjectMapCCP4: Current mean = -0.66 and stdev = 0.074981.

  ObjectMap: Map read.  Range: -0.511 to 0.616

  Crystal: Unit Cell  200  300  100

  Crystal: Alpha Beta Gamma90.000  100.354   90.000

  Crystal: RealToFrac Matrix

  Crystal:0.0060   -0.0.0011

  Crystal:0.0.0045   -0.

  Crystal:0.0.0.0053

  Crystal: FracToReal Matrix

  Crystal:  2000.  -34.5817

  Crystal:0.  3000.

  Crystal:0.0.  100

  Crystal: Unit Cell Volume  6993536.

  ExecutiveLoad: E:/ bdligand002.ccp4 loaded as bdligand002, through state 
1.

  PyMOLisomesh fo-fc_ligand, bdligand002, 3, ligand, carve=2

  Executive: object fo-fc_ligand created.

  Isomesh: created fo-fc_ligand, setting level to 2

  ObjectMesh: updating fo-fc_ligand.



  It seems like no error, but my ligand map, fo-fc_ligand has no density map 
shown up. I also tried to show the whole mesh at level 2.0 for bdligand002. I 
still could not see the density map.



  My pymol is version 1.3 in windows 8 operation system.  





  Any help will be greatly appreciated!



  Thanks in advance



  hongshi 

Re: [ccp4bb] identifying protein crystals via visible light only?

[Zhijie Li]

Hi Richard,

I am not sure if this is what you are looking for: second order nonlinear 
optical imaging of chiral crystals (SONICC).  It is not based on a 
computational algorithm  but the nonlinear optical property of chiral 
crystals to double the wavelength when illuminated by intense light.

Some recent publications:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3264792/
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3345893/
http://www.ncbi.nlm.nih.gov/pubmed/23765294

The instrument is also available on market.

Zhijie


-Original Message- 
From: Richard Gillilan

Sent: Tuesday, February 04, 2014 10:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] identifying protein crystals via visible light only?

Some years ago, I remember hearing about a microscope that used *visible* 
light combined with some proprietary image processing algorithm to
distinguish between protein crystals, salt, and background. I can't remember 
the company name or researchers involved.


Has anyone here heard of this?


Richard Gillilan
MacCHESS
Cornell 

Re: [ccp4bb] 3D stereo notebook

[Zhijie Li]

Hi Zhongzhou,

For laptops, the passive stereo (Zalman mode) would be the easiest way to go. 
All you need to do is to plug that monitor to the VGA port of you laptop. The 
halving of the vertical resolution under stereo mode only affects reading the 
characters, which can be solved by setting the fonts bigger.

I am not sure if LG D-2342 is still in market. But if not they should have some 
newer models of the same technology. 

Zhijie

From: zhongzhou chen 
Sent: Friday, November 22, 2013 9:33 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] 3D stereo notebook

Dear all,


I am looking into ordering a stereo notebook to view 3D protein structure.
Which notebook can you recommended? I require that the 3D notebook will be used 
with Linux and Windows, so support of the correspoding graphics card with both 
systems is required.

In addition,  I found that Dell XPS17XPS17D-428 can be used in windows. However 
I do not know whether 3D view is OK or not in Linux? Moreover, the displayer is 
17 inch, I think that it is a little larger.

Any comments or experience are welcome. 






Thanks and best wishes,


zhongzhou chen

china agricultural university

Re: [ccp4bb] AW: [ccp4bb] Dealnig with O-linked mannose

[Zhijie Li]

Hi Dimetry,

The difference between LINK and LINKR can't be explained better:
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg11865.html

Older versions of COOT used to not display the LINKR record, but now the
newer versions display both LINK and LINKR records as dotted bonds. I assume
that means COOT will now treat LINKR as LINK, otherwise there is no point
displaying a bond but not taking its meanings. However outside the
refmac-COOT universe, LINKR is not a legitimate record.

Zhijie


-Original Message- 
From: Dmitry Rodionov

Sent: Friday, November 22, 2013 11:11 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: [ccp4bb] Dealnig with O-linked mannose

Thanks, Herman.

Zhijie, what is the difference between LINK and LINKR?

Maybe this will help somebody (though phenix-related):
phenix.link_edits parses the pdb for LINK records and outputs the pdb.edits
file.
Alternatively, phenix.ligand_linking can guess what should be bonded in the
absence of LINK records and produce apply_link.def

Best rgards,
Dmitry

On 2013-11-22, at 3:18 AM, herman.schreu...@sanofi.com wrote:

Using your favorite editor, you can copy the LINK record from the pdb file 
generated by Coot and paste it into the pdb file produced by Phenix. You 
can also make a script to do this. This is what I did during the time LINK 
records were not properly handled by coot, refmac and buster.


Best regards,
Herman



-Urspr�ngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Dmitry Rodionov

Gesendet: Donnerstag, 21. November 2013 22:09
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Dealnig with O-linked mannose

Thank you all for helpful suggestions.

My question was how to properly connect a mannose to a serine and 
real-space refine the result. My apologies for not being clear enough.


Coot can't find MAN-SER or SER-MAN in it's library (Coot 0.7.1, mon_lib 
5.41)
It does not automatically make the bond between MAN C1 and OG of SER 
either.


Here is the way I finally made the connection followed by refinement it in 
Coot:


1) Get monomer... MAN
2) real-space refine MAN into reasonable position
3) Delete hydrogens and reducing hydroxyl
Bond is not detected
4) Extensions-Modelling ...-make Link (Click 2 atoms) ... Click on C1 of 
MAN and OG of SER

dashed bond appears
5) Sphere refine

A whole different issue that was touched in the replies is how this model 
will be handled by refinement programs.
For the sake of a record, I am using Phenix and it seems to not respect 
the described link without massaging by means of either pdb.edits or 
apply_link.def.
Also, phenix.refine does not produce LINK records in the output PDB, so 
step 4 might have to be repeated.


Best regards,
Dmitry 

Re: [ccp4bb] Dealnig with O-linked mannose

[Zhijie Li]

Hi Dmitry,

COOT does have the MAN-SER linkage record in its monomer lib, but it won't 
detect the bond for you. It also haven't provided an interface for the user 
to specify the bond type yet.
The COOT procedure you described is perfectly fine for generating a generic 
covalent bond record for a PDB file. However without specifying the linkage 
to be MAN-SER you do lose some restraints for the bond angles (as Engin 
pointed out in his post regarding phenix.refine).


It seems that refmac is the only program (that I've used so far) that will 
automatically detect the N- and O-glycan linkages and put that information 
into the header of a PDB file. This seems to be the easiest way of 
generating a proper LINKRMAN-SER line in the PDB header. Note that 
refmac writes a LINKR record for its own use, not LINK which is the PDB 
standard. You can simply manually change the LINKR to LINK  to turn it 
into a PDB standard record, but LINKR works fine for newer versions of COOT 
too.  The advantage of taking this path instead of writing a LINK line 
manually (or modifying the LINK line you get from COOT) is that you can be 
sure that the MAN-SER is put in the right columns - PDB format is 
position-sensitive.


Alternatively, here is a LINK line that you can modify to fit your sequence 
and paste to your PDB file. Just make sure you only replace characters, not 
inserting/deleting any:
LINK C1  MAN B1001 OG  SER B 912 
MAN-SER


For phenix.refine, yes, it does not write any linkage information to the 
header or use those information stored in PDB headers. But you can always 
paste that LINK line you get from other places to the PDB files generated by 
phenix.refine. There is no need to redo the building  in COOT every time. 
The LINK line only specifies the bonding between two atoms. So as long as 
you haven't renumbered the residues or changed the chain IDs, you can 
transfer the same LINK line among your PDB files of the same protein (i.e. 
you do not even have to remake the link in COOT for every structure of the 
same protein: just paste the LINK line and make sure the residue numbers are 
right).


Zhijie



-Original Message- 
From: Dmitry Rodionov

Sent: Thursday, November 21, 2013 4:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Dealnig with O-linked mannose

Thank you all for helpful suggestions.

My question was how to properly connect a mannose to a serine and real-space 
refine the result. My apologies for not being clear enough.


Coot can't find MAN-SER or SER-MAN in it's library (Coot 0.7.1, mon_lib 
5.41)

It does not automatically make the bond between MAN C1 and OG of SER either.

Here is the way I finally made the connection followed by refinement it in 
Coot:


1) Get monomer... MAN
2) real-space refine MAN into reasonable position
3) Delete hydrogens and reducing hydroxyl
Bond is not detected
4) Extensions-Modelling ...-make Link (Click 2 atoms) ... Click on C1 of 
MAN and OG of SER

dashed bond appears
5) Sphere refine

A whole different issue that was touched in the replies is how this model 
will be handled by refinement programs.
For the sake of a record, I am using Phenix and it seems to not respect the 
described link without massaging by means of either pdb.edits or 
apply_link.def.
Also, phenix.refine does not produce LINK records in the output PDB, so step 
4 might have to be repeated.


Best regards,
Dmitry

Re: [ccp4bb] Dealnig with O-linked mannose

[Zhijie Li]

It seem the LINK line I provided eariler was chopped by the email system.

Here it is again:
LINKRC1**MAN*C***1*OG**SER*B*912MAN-SER
Simply replace each * with a space and change the residue IDs.

Also to clarify the procedure of using refmac to generate the MAN-SER 
linkage line:
1 In COOT, get monomer-MAN, delete hydrogens, delete O1. Drag the MAN to 
the proper position. C1 has better be ~1.4A away  from OG and mind the 
anomer conformation.
It seems that refmac detects covalent bonds mainly based on the distances. 
If you accidentaly put the ligand too close to something it should not be 
linked to (happens when you have poor electron density for positioning the 
ligand), refmac might complain that a new ligands is found but the geometry 
information is not provided. In such case the error message is a little 
misleading to the user: it is a new covalent bond, not a new monoer that's 
not described.
2 Merge molecules, Save PDB. For linkages that can be found in the standard 
monomer lib, you do not need to make the bond in COOT. It is refmac that 
will add this record.

3 Send the saved PDB to refmac. Do restrained refinement.
4 Check the resulting PDB to see if you now have the LINKR...MAN-SER line.

And in the refmac log you can find this line:
WARNING : link:MAN-SER  is found dist = 1.178 ideal_dist= 1.439
  ch:BB   res: 912  SER  at:OG  .-CC   res:   1  MAN 
at:C1  .



Zhijie

-Original Message- 
From: Dmitry Rodionov

Sent: Thursday, November 21, 2013 4:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Dealnig with O-linked mannose

Thank you all for helpful suggestions.

My question was how to properly connect a mannose to a serine and real-space 
refine the result. My apologies for not being clear enough.


Coot can't find MAN-SER or SER-MAN in it's library (Coot 0.7.1, mon_lib 
5.41)

It does not automatically make the bond between MAN C1 and OG of SER either.

Here is the way I finally made the connection followed by refinement it in 
Coot:


1) Get monomer... MAN
2) real-space refine MAN into reasonable position
3) Delete hydrogens and reducing hydroxyl
Bond is not detected
4) Extensions-Modelling ...-make Link (Click 2 atoms) ... Click on C1 of 
MAN and OG of SER

dashed bond appears
5) Sphere refine

A whole different issue that was touched in the replies is how this model 
will be handled by refinement programs.
For the sake of a record, I am using Phenix and it seems to not respect the 
described link without massaging by means of either pdb.edits or 
apply_link.def.
Also, phenix.refine does not produce LINK records in the output PDB, so step 
4 might have to be repeated.


Best regards,
Dmitry

Re: [ccp4bb] Dealnig with O-linked mannose

[Zhijie Li]

Hi Dmitry,

I think the best way is not to make a new monomer. MAN-SER and MAN-THR 
linkages do exist in the ccp4 monomer lib. If you simply build a mannose 
with its O1 removed and put the C1 ~1.4 Angstrom to the OG of the serine I 
think refmac should be able to detect the linkage. When this happens, you 
should see a LINKR MAN-SER  line in the header of the resulting 
PDB file. COOT may not show a bond line for linkr record, but the real space 
refine should work fine.


If you need to refine the structure with phenix.refine then you need to make 
an edit file to specify that the mannose C1 is linked to the ser OG by a 
covalent bond.


Zhijie

-Original Message- 
From: Dmitry Rodionov

Sent: Wednesday, November 20, 2013 1:49 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Dealnig with O-linked mannose

Good day!

I am refining what appears to be O-mannosylated protein structure.

In my hands Coot (0.7.1) does not form the SER-MAN bond automatically.
I made a SER-MAN.cif with jLigand, which takes care of the glycosidic bond. 
However, now the peptide bonds are not made to this custom residue (same 
chain, consecutive numbering).


Am I doing something wrong? How can I fix this?

Many thanks,

Dmitry

Re: [ccp4bb] Staining Crystals with comassie

[Zhijie Li]

Hi Danilo and all,

A little trick for the glutaraldehyde staining: you can hang a 1-2uL drop 
of 25% glutaraldehyde (or the most concentrated stock solution you can find) 
besides your crystal drop in the vapour diffusion chamber. The 
glutaraldehyde will get into the crystal drop via vapour diffusion. The 
color will normally show within 2hrs and become very intense overnight. It 
is also a gentle way of crosslinking the crystals 
(http://scripts.iucr.org/cgi-bin/paper?wb0066, and 
http://hamptonresearch.com/tip_detail.aspx?id=74, ).
Care should be taken when handling aldehyde concentrates: do not breathe 
it, and do not let the vapour get in touch with your eyes. Waste can be 
inactivated by concentrated glycine solution.


BTW, the acetic acid in the coommasie blue solution seems unnecessary in a 
crystal staining solution. The solution recipe seems to be taken from a gel 
staining solution. When staining polyacrylamide gels, the acid (oringinally 
HCl) is supposed to denature the proteins so that they do not diffuse in the 
gel. The MeOH is for solubilizing the commonly used coommassie R250. 
(Another thing: I strongly suggest to substitute the MeOH in PAGE staining 
and de-staining solutions with EtOH. EtOH works perfectly fine, without 
MeOH's poisonous effect on human. Our staining solution contains 20% EtOH 
and 20%HAc.)
For staining crystals, we do not need to add the acetic acid. Also 
coommassie G250 is more soluble in water than the R250 by having methyl 
groups instead of ethyl groups. 0.5% coommassie G250 can be readily made in 
DMSO or 95% EtOH. Then this stock solution can be diluted with water or the 
mother liquor 10x-100x for the staining. Many crystals can tolerate up to 
10% DMSO.


Zhijie



-Original Message- 
From: Danilo Belviso

Sent: Wednesday, October 16, 2013 3:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Staining Crystals with comassie

Dear All,

izit dye is a solution containing methylene blue that you could prepare
in your lab. I usually prepare a solution of 0.05%w/v of dye in water
and then I add a volume of dye solution equals to 10% of the volume of
the drop containing the crystal to test. I prefer to add the dye
solution in small portions (if the volume permits) every 2-3h in order
to limit the shock due to the new solution on the crystal. You should
remember that this test is not definitive: the dye is a cationic dye,
that needs of anion counter part to bind the protein. Therefore, the dye
is not able to colour all protein crystals: in addition, colouration is
affect by pH of the crystallization condition, since low pH could
increase the positive charge on the protein reducing its ability to bind
the dye.

You could try also glutaraldehyde as alternative. In order to perform
this test, you should put the crystal into a low ionic strength buffered
solution containing up to 2% glutaraldehyde. In this condition,
formation of Schiff bases with the lysines and N-term residues occurs
and the crystal become a yellow gel, while salt crystals dissolve and
should not be coloured.

To perform comassie crystal staining you should prepare a solution of
1% comassie in 40% MeOH and 10% Glacial Acetic Acid and add this
solution as in methylene blue test. However, I rarely use this test
because the use of MeOH and Glacial Acetic Acid causes the crystal
dissolution.

Only a final tip: obviously these tests enable you to distinguish
between protein and salt, however they do not differentiate between
protein in the crystal and protein in solution. For these reason, in
some cases could be difficult to see the crystal colouration due to the
low contrast with the colouration of the solution. Hence, I prefer to
put the crystals to test in a new solution with the same formulation of
the drop where the crystals have grown but without protein and perform
here the dye test that I have chosen. In this way, you can easily see
the colouration of the crystal without background effect.

I hope I've helped you.

Danilo





On Tue, 15 Oct 2013 13:29:20 +0530, Swastik Phulera
swastik.phul...@gmail.com wrote:

Dear All,
I am looking for a method to quickly differentiate between salt and
protein crystals. I have been told  thats its a popular alternative
to the commercially available izit dye. I would appreciate if some one
would share their comassie crystal staining protocol.

Swastik 

Re: [ccp4bb] Staining Crystals with comassie

[Zhijie Li]

Izit is an aqueous solution of methylene blue, which you can prepare simply and 
cheaply. Look here: 

http://www.ysbl.york.ac.uk/ccp4bb/2000/msg00387.html

One word of warning: not every crystal stains. I found poking crystal with hair 
or glass fibre to see if it cracks or breaks is more reliable. Salt crystals 
react to attacks from glass fibres like rocks (as indeed what they are).


From: Swastik Phulera 
Sent: Tuesday, October 15, 2013 3:59 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Staining Crystals with comassie

Dear All,

I am looking for a method to quickly differentiate between salt and protein 
crystals. I have been told  thats its a popular alternative to the commercially 
available izit dye. I would appreciate if some one would share their comassie 
crystal staining protocol.


Swastik

Re: [ccp4bb] shape complimentarity for small molecule

[Zhijie Li]

Hi Tom,

Yes, SC can be used for calculating protein-small molecule ligand SC. Simply 
put the ligand and protein on two separate chains. You probably need to edit 
the $CLIBD/sc_radii.lib file to add some atom radii for your ligand. For 
example:


ABC  N*  1.65
ABC  C*  1.90

Zhijie


-Original Message- 
From: Brett, Thomas

Sent: Tuesday, September 10, 2013 4:56 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] shape complimentarity for small molecule

Hi all:
If have used SC previously to calculate shape complimentarity for 
macromolecular complexes. Can this also be used to calculate shape 
complimentarity for a (say) protein/small molecule inhibitor complex? Or is 
there some other metric/software that can/should be used to quantitate how 
well a small molecule fits a pocket?

thanks
-tom


Tom J. Brett, PhD
Assistant Professor of Medicine
Division of Pulmonary and Critical Care
Washington University School of Medicine
Campus Box 8052, 660 S. Euclid
Saint Louis, MO 63110
http://brettlab.dom.wustl.edu/ 

Re: [ccp4bb] off topic: good peak on gel filtration

[Zhijie Li]

Hi Ed,

I guess by 24mL SD200 Peter meant the Superdex 200 10/300 column, which 
most of us should be quite familiar with. According to GE healthcare, a new 
Superdex 200 10/300 GL column should have TP 25000/m. For comparison, a new 
Superdex200 16/600 PG, which uses bigger beads, has TP 13000/m. The TP 
difference of the two should be mainly caused by the different resin sizes.
Of course in reality columns change over time and in cases like Peter's, it 
might be a good idea to test the performance of the column before drawing a 
conclusion. When we are concerned about resolution of a column, we load a 
standard sample and calculate the TP based on the peak shape. As I remember, 
GE healthcare's SEC manuals has recommended procedures on TP determination.


Zhijie


-Original Message- 
From: Edward A. Berry

Sent: Saturday, June 29, 2013 7:43 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] off topic: good peak on gel filtration

Peter Hsu wrote:

Hi all,

I've generally always thought as long as the peak was symmetrical and not 
too broad would suggest a good sample. However, looking at my previous 
runs in the past, I've had peaks as narrow as 1.5-2mL on a 24mL SD200, or 
slightly broader peaks with about 3mL (all symmetrical peaks, roughly 
similar amounts loaded on the columns). I'm curious to see what people's 
views are as far as what constitutes a broad peak and how much that can 
end up affecting crystallization of the sample.


Thanks for any responses.

Peter

The width itself may not be a good indicator unless its always the same 
protein- in general a molecule that elutes later

will have a broader peak.
Supposing that each time a molecule diffuses into the stationary phase it 
resides there for a certain time on the
average, then the extra retention time is proportional to that time, times 
the number of times it enters stationary
phase (N, theoretical plates). The variance in elution time is 
proportional to the square root of N (like standard
error of the mean) and the dwell time. This gives sigma/(retention time) = 
1/sqrt(N). If N is the same for all
molecules, the criterion to look at is peak width divided by retention time. 
If it varies (the reason some molecules
elute slower is not just that they stay in the stationary phase longer, but 
also they enter more often; k-on as well as
k-off) that would still be better than just peak width.  People don't talk 
about theoretical plats and HTEP much any
more, perhaps because the driving force in chromatography is HPLC and FPLC, 
and fast chromatography is antithetical to

good resolution?

However I'm not familiar with this column and can't advise. You can 
calculate N more exactly (see wikipedia van Deemter
equation) as 8*ln(2)*square of (elution volume over width at half height), 
divide length of column by that to get HETP,
and compare with values like .7 mm reported for resins like ultragel A at 
optimum (very slow) flow rate.


eab 

Re: [ccp4bb] Negative FoFc around ligand

[Zhijie Li]

Hi Kavya,

If I understand you correctly (from title and text), you meant your negative 
FoFc was around your ligand, is that right? I wonder if this is a case in 
which the ligand has an occupancy below 1, but was modeled as 1, so the 
refinement program had to give it a high B factor to compensate that, which 
results in the electron density bleeding into nearby space where there 
should not be so much of it.
If you want to test this hypothesis, one thing you can try is to set the 
occupancy to 0.25, 0.5,0.75 and so on, and refine a few cycles to see what 
happens to the maps. Also, what's the B factor of the ligand, and what's the 
B of the nearby protein atoms? The difference between them can also give you 
some hint for guessing the ligand's occupancy. Some refinement 
programs(phenix.refine and shelx) can also let you refine the ligand's 
occupancy.


As to the missing atoms, that could be caused by alternative conformations 
of the ligand - assuming you have already done a thorough refinement.


Zhijie

--
From: Kavyashree Manjunath ka...@ssl.serc.iisc.in
Sent: Friday, May 24, 2013 12:50 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Negative FoFc around ligand


Dear users,

I am using refmac 5.7.0029 for refining a structure (resolution 2.2 Ang)
bound to 2 ligands. After MR There is a very clear density of ligands but
after refinement, I get negative fofc map near one of the ligand upto 5
sigma. However its 2fofc map covers the whole ligand. Also for the other
ligand, I do not see any 2fofc density (at 3 sigma) for 2 atoms, without
these atoms the ligand is unrealistic. But the density comes up around
these at around 0.7 sigma.
Overall completeness is 99.9%
Rmerge  7.5%

What else I need to check in the data. Kindly provide some suggestions.

Thanking you
Regards
Kavya





--
This message has been scanned for viruses and
dangerous content by MailScanner, and is
believed to be clean. 

Re: [ccp4bb] Off-topic (SPR)

[Zhijie Li]

Hi Jahan,

Maybe you are using a new model of BiaCore.  Our old BiaCore X never cares to 
tell us how to do our experiment. However if your RU did go below that of the 
empty chip, then maybe you really didn't get anything conjugated.

The pH 3.8 condition is not good for the amine coupling reaction. The reaction 
will be very inefficient when pH drops below 5. You can try conjugating at 
higher pH, such as 6, 5.5 or 5. If you can not get protein adsorbed to the 
surface by electrostatic attraction, then simply increase the protein 
concentration - 0.05, 0.1mg/mL, or as much as you can afford to use, and extend 
the time of reaction (you can slow down or even stop the flow of protein to get 
some incubation). You should see at least 100 or 200 RU increase after a 
successful coupling for your 15kD protein. 

How many Lys do you have in the 15kDa protein? If you have very few Lys, and 
the protein is made in E coli, you can add a short poly Lys tail to either ends 
of the protein. This also has an additional advantage if some of the Lys 
residues are located in the binding surface.

Zhijie


From: Jahan Alikhajeh 
Sent: Wednesday, May 08, 2013 3:08 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Off-topic (SPR)


Dear pals,

Sorry for my off-topic question but its been a while I am challenging to get a 
15 kDa protein coated on CM5 chip. Calculated PI of protein is 5.9  I tried 
Acetate buffer pH5.2 and 3.8 for coating under normal procedure of Biacore. 
After immobilization I saw this message: response level decreased! This 
happened for me several times. Does anyone have any idea what's going on? 

Cheers,
Jahan


 

Re: [ccp4bb] off topic, BN PAGE

[Zhijie Li]

Hi Careina,

Gels certainly are convenient and powerful tools when they work. For normal 
native gel, do not forget that if you are using the Laemmlli buffer system (ie. 
tris-glycine), the actual pH during running is pH 9 and the ionic strength 
changes over time. There are other buffer systems, but you still need to be 
careful with the results. 
The other consideration is that when running a dimer in gel, if the dimer is 
not very stable, during a long run (for example, regular native gel often take 
hours), the dimer can dissociate significantly, resulting in a smear or total 
loss of the dimer band. 

I think if you can run gel-filtration experiments(a little more expensive in 
terms of equipment and protein consumption), you probably can draw conclusions 
with more confidence due to: 1) you can see the peaks in 20 minutes so even if 
the dimmer dissociates it will dissociate less in less time; 2) you can 
equilibrate the column in your buffers so there is no issue about the real 
condition you are testing. The drawback is that you can't test your different 
conditions in parallel. But with gels, your conditions only exist in the 
loading wells, when the proteins enter the gel and spend the 1-5 hrs there, 
your initial conditions are lost anyways.

Zhijie


From: Careina Edgooms 
Sent: Thursday, March 28, 2013 3:03 AM
To: Zhijie Li 
Subject: Re: [ccp4bb] off topic, BN PAGE


Hi Zhijie


Thanks for the helpful reply. The attractive thing about BN PAGE (if it works 
of course) is that it is so quick, inexpensive and simple to perform. I am 
working with a protein that exists in the native state as a mixture of monomer 
and dimer. I wish to monitor the effects of various things on this monomer 
dimer equilibrium. The simplest way of doing this is to run a gel at the end of 
the experiment and quantify the bands with densitometry to check the effect on 
the monomer dimer equilibrium.


When I run this protein on a BN PAGE gel it runs as 2 bands. I initially 
assumed these 2 bands to be a true representation of the monomer dimer 
equilibrium but now I am wondering if the coomassie could actually interfere 
with the contacts so that even if the protein was in a state where it was 
entirely dimeric, it would appear on the gel as 2 bands because the coomassie 
is interfering with the dimer interface. What I'm attempting to do now is run a 
regular native page without coomassie. Because my protein is very basic this 
means I will have to swap the electrodes. If this also shows 2 bands then I 
wonder if it is safe to assume that the BN PAGE works and coomassie does not 
interfere?


Careina




From: Zhijie Li zhijie...@utoronto.ca
To: Careina Edgooms careinaedgo...@yahoo.com 
Cc: CCP4BB@JISCMAIL.AC.UK 
Sent: Thursday, March 28, 2013 1:28 AM
Subject: Re: [ccp4bb] off topic, BN PAGE



Hi Careina,

BN PAGE can be affected by many factors. Considering the complexity and the 
chance of winning and the amount of information you gain even when you win, I 
do not recommend fighting it. 
BN PAGE, like other gel-based methods, requires that your complex is fairly 
stable - not having a weak affinity and not a high dissociation rate. Also the 
complex formation should be compatible with the gel running condition: the pH 
and salts and other things. Coomassie itself may compete for some hydrophobic 
surfaces or positively charged residues on the proteins - in such case there's 
little you can do. 
If you see a band corresponding to the expected complex weight, then 
congratulations, BN gel might be your tool (with cautions though as you can 
also get false positives with BN gel). But if not, then my suggestion is to 
move to other techniques such as gel filtration, analytical 
ultracentrifugation, BiaCore, ITC and so on. I had a case in which I could 
confidently show complex formation with gel-filtration and Biacore, but not 
with BN gel.

Zhijie


From: Careina Edgooms 
Sent: Wednesday, March 27, 2013 5:24 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] off topic, BN PAGE


Hi


Has anyone found the coomassie in a BN PAGE to be interfering with the 
oligomeric structure of their protein? If so, how did you deal with this?
Thanks


Careina

Re: [ccp4bb] ./configure problem

[Zhijie Li]

Hi Alex,

The CCP4 package you downloaded seems to be a prebuilt binary package for the 
X86_64 architecture. You do not need to compile yourself - and you can't 
without the source code. Running ./configure is for configuring the compilation 
environment before running make to compile source codes, which dose not apply 
to binary packages.

I guess you probably was following this page: 
http://www.ccp4.ac.uk/dist/INSTALL.html. The information on that page is for 
compiling the source code, not for the binary packages. The layout of that page 
is a little confusing: it does mention how to download the binary packages and 
started to say then first you need to decide where you want to put the 
software, but then it suddenly jumps to how to build the CCP4 from source 
code. I think this might be something the authors want to fix.

For prebuilt binary packages, please take a look at the INSTALL file in the 
unpacked CCP4 directory for installation instructions. The CCP4 binary 
installation is very simple: run the BINARY.setup in the unpacked CCP4 
directory. After it is done, append this line to /etc/bashrc (assuming your 
default shell is bash):

source /path_to_ccp4/ccp4.setup-sh

Then open a new shell, type ccp4i, you should see the CCP4 user interface.

Zhijie





From: A K 
Sent: Tuesday, March 26, 2013 8:16 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] ./configure problem


Hi there,
I am trying to install ccp4 on a scientific linux system. I've got stuck in the 
step where I need to configure the system. After sourcing ccp4.setup, I go to 
the main ccp4 folder and try ./configure help, but I get the error bash: 
./configure: No such file or directory. It looks as if the configure script 
has not been included in my tar file. The file I unzipped was called 
ccp4-6.3.0.1-arp-linux-x86_64.tar.gz. Any suggestion is highly appreciated.
Alex

[ccp4bb] COOT running slow after upgrading to CENTOS 6.4 from 6.3

[Zhijie Li]

Hello,

We have a curious situation here: after upgrading CentOS 6.3 to 6.4, COOT runs 
slowly every time after the go to atom command is executed - every rotation 
of the molecules takes nearly 1 second to finish. But if the go to atom 
command is never sent then the speed is normal. A COOT running in a virtual 
machine windows XP on the same computer behaves OK though. It doesn't seem to 
be a conflict with the CentOS 6.4 either, because COOT on another computer with 
newly installed CentOS 6.4 runs perfectly fine. So I guess it might has to do 
with some libraries that COOT might use. The computer with trouble have python 
upgraded to 2.7 from the python source code when it was running CentOS 6.3, 
could this be a problem?

Zhijie

Re: [ccp4bb] column tube

[Zhijie Li]

Hi George,

What is the plastic tube? Is it part of the end adaptors or it is part of the 
tubing? What type of plastic it is? If you can provide a photograph of the 
broken part maybe someone can give you more useful suggestions. 

Zhijie


From: George Kontopidis 
Sent: Wednesday, March 20, 2013 2:28 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] column tube


Dear Colleagues,

 

Apologies for the non-crystallographic question but it very likely that someone 
out there may have the answer.

I have a  chromatography column. Unfortunately the company does not provide a 
replacement for the plastic tube, which should not cost more than 100 €. The 
cost for a new column is more than 1500 €!

Does anybody know if there is a company that could provide custom-made plastic 
tubing for chromatography columns?

 

Thanks in advance for your answers,

 

 

George Kontopidis

 

 

Assοciate Professor of Biochemistry

Veterinary School, University of Thessaly

Karditsa 43100, Greece

Tel: +30 24410 66017

Mob: +30 69 342 643 75

e-mail: gkontopi...@vet.uth.gr

web site: http://www.vet.uth.gr/english/

 

Re: [ccp4bb] column tube

[Zhijie Li]

Hi George,

I think that part can be made from an aluminum or a steel tube with proper OD. 
The ID seems only need to be large enough to allow the glass tube to fit in - 
depending what is allowed by the two black end adaptors. You can check your 
engineering department or some machine shops to find some one to cut the tubes, 
ground the ends and machine the threads for you. Of course you won't be able to 
see through the wall now, but it will be a very strong column. The expense I 
think would mainly be labor.

Zhijie


From: George Kontopidis 
Sent: Wednesday, March 20, 2013 3:27 PM
To: 'Zhijie Li' 
Subject: RE: [ccp4bb] column tube


Dear Zhijie, 

 

 

I am looking for the external clear plastic tube of   

Bio-rad UNO Q1 Column  720-0001

 

Column information:

http://www.bio-rad.com/prd/en/GR/LSR/PDP/d825a6ab-49cf-4140-91c3-ea131c886b74/UNO-Monolith-Anion-Exchange-Columns

 

 

see also attached the broken plastic tube.

 

Unfortunately the company does not provide me with additional information 
regarding plastic type. Although we know that the plastic should stand 700 psi 
pressure.

 

thanks

 

George

 

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Zhijie Li
Sent: Wednesday, March 20, 2013 9:13 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] column tube

 

Hi George,

 

What is the plastic tube? Is it part of the end adaptors or it is part of the 
tubing? What type of plastic it is? If you can provide a photograph of the 
broken part maybe someone can give you more useful suggestions. 

 

Zhijie

 

From: George Kontopidis 

Sent: Wednesday, March 20, 2013 2:28 PM

To: CCP4BB@JISCMAIL.AC.UK 

Subject: [ccp4bb] column tube

 

Dear Colleagues,

 

Apologies for the non-crystallographic question but it very likely that someone 
out there may have the answer.

I have a  chromatography column. Unfortunately the company does not provide a 
replacement for the plastic tube, which should not cost more than 100 €. The 
cost for a new column is more than 1500 €!

Does anybody know if there is a company that could provide custom-made plastic 
tubing for chromatography columns?

 

Thanks in advance for your answers,

 

 

George Kontopidis

 

 

Assοciate Professor of Biochemistry

Veterinary School, University of Thessaly

Karditsa 43100, Greece

Tel: +30 24410 66017

Mob: +30 69 342 643 75

e-mail: gkontopi...@vet.uth.gr

web site: http://www.vet.uth.gr/english/

 

Re: [ccp4bb] off topic question BIAcore problem

[Zhijie Li]

Hi Xianchi,

First of all: a very slow dissociation rate can also be an artifact: the 
analyte can be simply precipitating on the surface. You do need to rule this 
out by proper controls. 

But there is no rule saying that 10e-6 s-1 off rate is not realistic. Even with 
protein-small molecule binding, one can get extremely slow dissociation if the 
interaction is very strong. For example, the dissociation of biotin from 
streptavidin has a rate constant of the 10e-6 s(-1) order. A very slow 
dissociation rate is often (if not always) correlated with very tight binding 
(for example KD in picomolar range or even smaller). What is your calculated 
KD? The binding phase of the BIAcore curves should also reflect the fact that 
the off rate is low (the Kon-obs has an off rate term).

We have measured some pico molar bindings with BIAcore in the past. It is 
doable, but difficult. You may also want to consider in-solution methods (so 
that you do not need to worry about artifacts caused by the surface), such as 
ITC (by competition method), or fluorescence-based methods. 

For BIAcore, when working with very strong bindings (KD 100pM), there are a 
few things to consider:
1) You may find great difficulties regenerating the chip - probably the biggest 
concern with BIAcore (considering the ridiculous price of chips).
2) How much RU should you conjugate to the chip? With strong interactions, as 
low as possible amount of your ligand should be labeled on the chip, for 4 
purposes: a) to make regeneration easier; b) to reduce rebinding effect; c) to 
reduce mass transfer effect; d) to make sure you do not take away significant 
amount of analyte from the solution (discussed in 3)).
3) If you plan to span the KD range with the analyte, then if the KD is in 
picomolar molar range, you are supplying the surface with extremely dilute 
analyte solutions. In such case, you need to calculate if your flow rate is 
high enough, to compensate the loss of solute due to binding to surface, 
otherwise the real concentration of the analyte in the mobile phase will be 
much lower than the assumed analyte concentration.
4) The association phase of the BIAcore experiment is also affected by the 
dissociation rate.  The observed binding rate Kon-obs contains a Koff term. 
When you are loading the analyte at near KD concentrations, the binding will 
take similar amount of time as the dissociation phase to reach plateau. The 
Kon-obs also contains a concentration terml, so the time required for reaching 
plateau will be shorter and shorter when you load with higher concentrations of 
the analytes.
5) The only proper way of labeling chip for slow dissociations is by covalent 
means.

HTH,

Zhijie




From: xianchi dong 
Sent: Wednesday, February 20, 2013 12:03 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] off topic question BIAcore problem


Dear all, 


Recently I have measure a set of kinetic data of a receptor ligand interaction 
using BIAcore 3000. To my surprise, the dissociation rate is very low (~ 
10e-6). During the measurement, I use a long dissociation time (2 hours) .I 
repeat several time which give very similar results. So I am wondering if the 
BIAcore can measure such a low off-rate kinetic. What is the limitation of 
BIAcore? Any review about that?


Thanks in advance.


Xianchi Dong
Research Fellow 
Children's Hospital Boston
Harvard Medical School

Re: [ccp4bb] off topic question BIAcore problem

[Zhijie Li]

Hi Xianchi,

How did you treat the control flow cell? And what is the composition of the 
control buffer that you use to disrupt binding? Is it denaturing or a mild 
condition? 

If the Rmax=150 and Koff=1e-6, then at 7200 s (2 hr), the signal will only drop 
1RU from 150RU (assuming you can reach the Rmax), is that what you see on the 
dissociation curve? In reality, when loading analyte at 100nM, assuming you 
have Kon=1e4, Koff=1e-6, binding for 30min would only let you reach ~125 RU, 
and at the lower concentrations will be much worse. Consequently the total 
dissociation after 7200s would be less than 1 RU on most curves - I am not sure 
if the Biacore 3000 will have a stable enough baseline for you to confidently 
measure that. If you try to fit the dissociation phase alone to get the Koff, 
the reported Koff won't be too accurate due to the low S/N ratio. On the other 
hand the 5-100nM spanning would generate significant changes on the binding 
phase, thus the global fitting should be able to get a relatively accurate KD. 
0.2nM from SPR and 1nM from in-solution experiment also sounds reasonable.

Zhijie



From: xianchi dong 
Sent: Wednesday, February 20, 2013 5:45 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] off topic question BIAcore problem


Thanks a lot for the kind reply. 


I used CM5 chip at pH 7.4. I am not quite sure about the PI of my protein but I 
have different truncations of my protein which behaved similarly. 
I can fit my data quite good with the Chi2 around 1 and Rmax 150. The kon is 
about 10e4 and the KD is around 0.2 nM. The concentration of analytes I used 
was from 100 nM to 5 nM. I  used a control buffer which can disrupt the 
binding. In that buffer, no binding was observed. 


I also used fluorescence anisotropy to measure the Ki of the interaction which 
showed a 1 nM binding compared with the 0.2 nM binding in the SPR assay. 



On Wed, Feb 20, 2013 at 12:03 PM, xianchi dong dongxian...@gmail.com wrote:

  Dear all, 


  Recently I have measure a set of kinetic data of a receptor ligand 
interaction using BIAcore 3000. To my surprise, the dissociation rate is very 
low (~ 10e-6). During the measurement, I use a long dissociation time (2 hours) 
.I repeat several time which give very similar results. So I am wondering if 
the BIAcore can measure such a low off-rate kinetic. What is the limitation of 
BIAcore? Any review about that?


  Thanks in advance.


  Xianchi Dong
  Research Fellow 
  Children's Hospital Boston
  Harvard Medical School

Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!

[Zhijie Li]

Hi Jacob,

Interesting topic.

This reminds me the posters I saw on ACA 2010, on the femto-second infrared 
laser based instrument . That instrument utilizes the nonlinear optical 
properties of  crystals of chiral molecules to detect very small crystalline 
materials from amorphous background: the crystals will double the frequency of 
the laser, turning the infrared light to visible light. I cannot recall the 
exact name of the technology now, unfortunately. 

Your case of observing in vivo GFP crystals is a little special in that the 
crystals are fluorescent. I guess if we scan cells over-expressing proteins 
with the above mentioned instrument, we might find that many proteins will do 
the same in cells. 

Naturally occurring in vivo crystals are not very rare. If we do not restrict 
the topic to proteins, then it is well known that many viruses readily 
crystallize in the host cell's nuclei and the resulting crystals or crystalline 
arrays can be observed under EM. And if we do not restrict the cells to 
mammalian cells, then there come the famous BT crystals. 

In addition, I just did some internet search and here are some interesting 
results:

1) Viral protein crystals can form in HEK cells infected by adenovirus 
(http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002894)
2) Bacterial infection can cause the infected epithelial cells to form 
pathological crystal-containing inclusion bodies in the cytosol 
(http://www.ncbi.nlm.nih.gov/pubmed/8940763).
3) Crystalline inclusion bodies are found in rabbit embryos 
(http://dev.biologists.org/content/44/1/31.full.pdf) and epididymis of the 
nine-banded 
armadillo(http://www.sciencedirect.com/science/article/pii/S0022532073800073). 
Actually if google crystalline inclusion body, there will be tons of 
literatures.
4) IgG crystallized in the ER when over expressed from a highly optimized CHO 
expression system (http://www.jbc.org/content/286/22/19917.abstract). This is 
particularly interesting as we know that whole IgGs are not so prone to 
crystallize, although the author do state that Crystallizing propensity was 
due to the intrinsic physicochemical properties of the model IgG.


Given the prevalence of in vivo crystallization, especially considering their 
correlation with inclusion bodies, I think it is reasonable to suspect that 
there are some cases that the inclusion bodies formed during over expression of 
transgenic proteins in E. coli are crystalline. I expect that we will be 
enlightened on this issue by somebody on the BB soon.

Zhijie




From: Jacob Keller 
Sent: Friday, February 15, 2013 2:44 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Sighting of Protein Crystals in Vivo?!


Dear Crystallographers, 


I was looking at some live, control HEK cells expressing just eGFP, and to my 
great surprise, saw littered across the dish what appeared to be small 
fluorescent needles (see attached--sorry about the size, but it's only ~1MB 
total.) Can these possibly be fortuitous protein crystals? They were too small 
to mount I think, and for what it's worth, parallel-transfected HeLa cells did 
not have these things. But, some needles could be seen in the DIC images as 
well, and the needles were only fluorescent with GFP filter sets, and not CFP, 
YFP, or texas red filters. I thought of whale myoglobin crystallizing on the 
decks of ships, but never thought I would see this


Jacob


-- 
***
Jacob Pearson Keller, PhD
Postdoctoral Associate
HHMI Janelia Farms Research Campus
email: j-kell...@northwestern.edu
*** 

Re: [ccp4bb] GeForce Graphics cards

[Zhijie Li]

Hi,

Since it is mentioned, I would like to take this chance to clarify:

We have used the Zalman and LG 2342P under Linux, Windows(XP and 7) and a VM 
Windows within a Linux system, on either desktop (with a minimal on-board 
graphic chip) or laptops. The monitor is quite plug-and-play in all cases we 
have tested. According to the internet, these monitors also work with Macs for 
stereo game play and stereo video viewing. 

In regard to the crystallographic software, the Zalman mode is supported by 
COOT, Chimera, and the new Pymol (but not the 0.99) - we have tested them all 
in both Linux and Windows. 

Passive 3D only requires the graphic program to send out the left and right 
images in alternating lines. To my understanding this suggests that it is 
independent of OS or graphic cards. But for Mac users, I think the first thing 
to check is whether the Mac COOT and Chimera has a Zalman mode option. 

Here's the link to my previous reply on the BB regarding our experience with 
the Zalman and LG D2342:
http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg28113.html

Zhijie




From: S K 
Sent: Wednesday, February 13, 2013 3:37 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] GeForce Graphics cards


Harry, 

I found this on pymol wiki NVidia 3D NVision kit only supports DirectX 
software for GeForce (gaming cards) on Windows; users are reporting that they 
are not able to run PyMOL with NVision with these cards. Get a newer model low 
end quadro ( G8x graphics core) without the 3 pin mini din (e.g. Quadro 370) 
or with the 3 pin mini din (e.g. Quadro 3700) for Windows. and this too  The 
GeForce cards do not support windowed openGL stereo, so we do not support these 
series of cards for the NVision 3D solution. For linux, you must have a quadro 
card that has a 3 pin mini din connector. The cheapest/oldest card that will 
work with linux is the Quadro 3700. WARNING: The Quadro FX1400 does not support 
3d vision stereo on Windows7 or Linux.   This was posted there a while ago and 
I was wondering if there has been any update to that which has not been posted.

Regarding the passive monitors, I think they are compatible with linux, after 
all we have been using zalman for long time, which is technically a passive 3D 
monitor. I also found posts on the ccp4bb from people happily using LG D2342P 
monitor, though linux or windows was not mentioned.

A 


On Wed, Feb 13, 2013 at 3:21 PM, Harry Mark Greenblatt 
harry.greenbl...@weizmann.ac.il wrote:

  BSD

  Dear Alex,


I'm not sure why you think the new Geforce cards will be an issue.  Please 
clarify.

  As far as stereo is concerned, Geforce cards only give stereo under Windows, 
not Linux.  If you want stereo under Linux you need the Quadro cards with the 
stereo option (for example, Quadro 4000 or 5000).  Not all Quadro cards support 
this option, and so would only give stereo under Windows, like the Geforce 
cards.

  Not sure about the passive type of stereo;  I have a feeling that will also 
only work under Windows.  I'm also not sure it works with Pymol and Coot.  
Perhaps someone can clarify this.

  As far as I know the stereo under Linux is only the active variety, using the 
NVidia 3D Vision glasses.

  Harry


  
  From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Alex Kavian 
[alek6...@gmail.com]
  Sent: Wednesday, February 13, 2013 9:47 PM
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: [ccp4bb] GeForce Graphics cards


  Hi there,

  I have an off-toptic question about Graphics card. My searches on pymolwiki 
and ccp4bb archives resulted in the following conclusion: Coot and pymol are 
not compatible with the new GeForce graphics cards. Hoewver, most of the posts 
I found were from 2009 and 2010. Does anyone here have any experience with 
GeForce 660 or 680 for stereo applications of Coot, Pymol or UCSF chimera? Will 
quadro cards work equally smooth in the stereo mode of these programs? Do 3D 
applications put too much pressure on the Graphics card which would justify 
installing dual Graphics card? (not sure if is relevant, but just for the 
record, we want to buy passive 3D monitor due to the budget restrictions).

  Thanks,
  Alex

Re: [ccp4bb] GeForce Graphics cards

[Zhijie Li]

Hi Alex,

The graphical computational power of even the lowest-end graphic chips these 
days will suffice the displaying of our models. To give you some idea: we 
are still using a Quadro FX 1000 card and an old CRT on one of our stereo 
systems; my 6-year old laptop has an ATI Radeon X1300, which can still drive 
the stereo display on Zalman or LG D2342P. You can check the benchmarks of 
these chips to see why dual card is guaranteed to be a waste.


If you are determined to go the passive 3D monitor(Zalman or LG D2342) path, 
then you actually do not need to worry about the graphic card - even the 
on-board one will do. Quadro cards are for the use with 120Hz monitors and 
shutter glasses, whereas the passive 3D monitors are CPL-based and do not 
need special graphic cards.


Having said all the above, UCSF Chimera might become a little demanding when 
displaying electron densities, so a discrete mainstream Nvidia or ATI chip 
would probably make life easier in a few cases.


Zhijie

--
From: Alex Kavian alek6...@gmail.com
Sent: Wednesday, February 13, 2013 2:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] GeForce Graphics cards


Hi there,

I have an off-toptic question about Graphics card. My searches on 
pymolwiki and ccp4bb archives resulted in the following conclusion: Coot 
and pymol are not compatible with the new GeForce graphics cards. Hoewver, 
most of the posts I found were from 2009 and 2010. Does anyone here have 
any experience with GeForce 660 or 680 for stereo applications of Coot, 
Pymol or UCSF chimera? Will quadro cards work equally smooth in the stereo 
mode of these programs? Do 3D applications put too much pressure on the 
Graphics card which would justify installing dual Graphics card? (not sure 
if is relevant, but just for the record, we want to buy passive 3D monitor 
due to the budget restrictions).


Thanks,
Alex 

Re: [ccp4bb] GeForce Graphics cards

[Zhijie Li]

Hi Alex,

We actually didn't invest on a discrete video card for the LG-D2342P system. 
The one we are using on the LG-D2342P desktop is the integrated Intel HD 
graphic 2000 chip, which is physically located in the CPU. The CPU is i7-2600. 
We installed a Linux OS on this desktop and also run a VirtualBoxed Windows XP 
inside the Linux for office jobs. Due to some limitations of the Virtualbox, 
the VM Windows system does not get hardware 3D acceleration, but still the 
speed of rendering is fine in most cases even for UCSF Chimera. In Linux there 
is no issue on speed.

Zhijie



From: A K 
Sent: Wednesday, February 13, 2013 6:51 PM
To: Zhijie Li 
Cc: CCP4BB@jiscmail.ac.uk 
Subject: Re: [ccp4bb] GeForce Graphics cards


Hi Zhijie,

thanks for the link. I was actually referring to that post. Can you please tell 
me what graphics card you are using?

Alex


On Wed, Feb 13, 2013 at 6:16 PM, Zhijie Li zhijie...@utoronto.ca wrote:

  Hi,

  Since it is mentioned, I would like to take this chance to clarify:

  We have used the Zalman and LG 2342P under Linux, Windows(XP and 7) and a VM 
Windows within a Linux system, on either desktop (with a minimal on-board 
graphic chip) or laptops. The monitor is quite plug-and-play in all cases we 
have tested. According to the internet, these monitors also work with Macs for 
stereo game play and stereo video viewing. 

  In regard to the crystallographic software, the Zalman mode is supported by 
COOT, Chimera, and the new Pymol (but not the 0.99) - we have tested them all 
in both Linux and Windows. 

  Passive 3D only requires the graphic program to send out the left and right 
images in alternating lines. To my understanding this suggests that it is 
independent of OS or graphic cards. But f
  or Mac users, I think the first thing to check is whether the Mac COOT and 
Chimera has a Zalman mode option. 

  Here's the link to my previous reply on the BB regarding our experience with 
the Zalman and LG D2342:
  http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg28113.html

  Zhijie




  From: S K 
  Sent: Wednesday, February 13, 2013 3:37 PM
  To: CCP4BB@JISCMAIL.AC.UK 
  Subject: Re: [ccp4bb] GeForce Graphics cards


  Harry, 

  I found this on pymol wiki NVidia 3D NVision kit only supports DirectX 
software for GeForce (gaming cards) on Windows; users are reporting that they 
are not able to run PyMOL with NVision with these cards. Get a newer model low 
end quadro ( G8x graphics core) without the 3 pin mini din (e.g. Quadro 370) 
or with the 3 pin mini din (e.g. Quadro 3700) for Windows. and this too  The 
GeForce cards do not support windowed openGL stereo, so we do not support these 
series of cards for the NVision 3D solution. For linux, you must have a quadro 
card that has a 3 pin mini din connector. The cheapest/oldest card that will 
work with linux is the Quadro 3700. WARNING: The Quadro FX1400 does not support 
3d vision stereo on Windows7 or Linux.   This was posted there a while ago and 
I was wondering if there has been any update to that which has not been posted.

  Regarding the passive monitors, I think they are compatible with linux, after 
all we have been using zalman for long time, which is technically a passive 3D 
monitor. I also found posts on the ccp4bb from people happily using LG D2342P 
monitor, though linux or windows was not mentioned.

  A 


  On Wed, Feb 13, 2013 at 3:21 PM, Harry Mark Greenblatt 
harry.greenbl...@weizmann.ac.il wrote:

BSD

Dear Alex,


  I'm not sure why you think the new Geforce cards will be an issue.  
Please clarify.

As far as stereo is concerned, Geforce cards only give stereo under 
Windows, not Linux.  If you want stereo under Linux you need the Quadro cards 
with the stereo option (for example, Quadro 4000 or 5000).  Not all Quadro 
cards support this option, and so would only give stereo under Windows, like 
the Geforce cards.

Not sure about the passive type of stereo;  I have a feeling that will also 
only work under Windows.  I'm also not sure it works with Pymol and Coot.  
Perhaps someone can clarify this.

As far as I know the stereo under Linux is only the active variety, using 
the NVidia 3D Vision glasses.

Harry



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Alex Kavian 
[alek6...@gmail.com]
Sent: Wednesday, February 13, 2013 9:47 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] GeForce Graphics cards


Hi there,

I have an off-toptic question about Graphics card. My searches on pymolwiki 
and ccp4bb archives resulted in the following conclusion: Coot and pymol are 
not compatible with the new GeForce graphics cards. Hoewver, most of the posts 
I found were from 2009 and 2010. Does anyone here have any experience with 
GeForce 660 or 680 for stereo applications of Coot, Pymol or UCSF chimera? Will 
quadro cards work equally smooth in the stereo mode of these programs? Do 3D

Re: [ccp4bb] 3D Monitors

[Zhijie Li]

Hi Sabine,

We are absolutely happy with our LG D2342P. The major advantages include the 
simplicity in setting up and the light weight of the glasses.


For more information, please read this thread:

http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg27816.html


Zhijie

--
From: Sabine Schneider sabine.schnei...@cup.uni-muenchen.de
Sent: Thursday, January 24, 2013 11:13 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] 3D Monitors


Hello everyone,

I know there already has been discussion about 3D monitors on the 
Coot/CCP4bb.
However since there are a few more out there now and I am currently 
thinking about buying one, I thought to get a few opinions from 
crystallographers would be nice! Especially if there are people happily 
model building with the cheaper LG 3D monitors? :-)


 So I am looking at the moment at:
- Zalman ZM-M215W 21,5in
- Zalman ZM-M240W 24in
- Samsung SyncMaster S27A750D 27in
- LG D2342P 23in / LG D2542P 25in

Thanks a lot!

Sabine 

Re: [ccp4bb] Boveral in SFCheck

[Zhijie Li]

Hi,

Seems not officially retracted from Nature either. On the paper's web page, 
there was only a line in small font read like this: 


There is a Brief Communications Arising (9 August 2007) associated with this 
document.

It took me more than half an hour to find this line. I normally won't read any 
line above the title. Now it proves to be a bad habit.

I am still trying to find this line in the PDF.

Zhijie




From: Michael Hadders 
Sent: Friday, December 14, 2012 2:57 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Boveral in SFCheck


Hi,

2HR0 I would stay far away from that one! It is a made up model, not based 
on any real data. Unfortunately, for reasons unclear to me, this structure has 
still not been retracted from the PDB. This B factor could just be a figment of 
the senior authors imagination

https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind0912L=CCP4BBD=0P=88327

Regards,

Michael


On Fri, Dec 14, 2012 at 3:34 AM, Eric Williams ericwilli...@pobox.com wrote:

  Please pardon me if this is a dumb question with an obvious answer... 


  I'm parsing SFCheck's plain text output as part of my dissertation, and I'm 
having trouble identifying one of the values. There are three overall B-factor 
values reported, one based on the Patterson origin peak, one based on the 
Wilson plot, and one that remains a mystery to me. Here's the relevant line 
(from 2HR0) with some lines before and after for context:


 R_stand(I) = sig(I)/I :0.397
 Number of acceptable reflections:  194123
 for resolution :  45.33 -  2.26
 Optical Resolution:   1.80
 Boveral,Effres,Padd:   40.751   2.032 777.887
 Expected Optical Resolution for complete data set:   1.80
   / Optical resolution - expected minimal distance between
 two resolved peaks in the electron density map./
 Resmax_used(opt):  2.26


  The mystery value is Boveral. I've found no explanation for it in either the 
SFCheck manual or the original journal article. Perhaps I'm missing something 
obvious, but someone would really make my day if they could point me in the 
right direction. Thanks! :) 


  Eric

Re: [ccp4bb] CCP4superpose_only superpose interesting residues

[Zhijie Li]

Hi Wenhe,

Superpose allows you to specify residue (as well as Ca/main chain/side chain) 
ranges. Also in COOT you can select the range of residues used in the LSQ 
superpose calculation. 

For superposing parts of the proteins only, a little trick is to cut the 
interested fragments from the original PDBs and make PDB files that contain the 
fragments only (simplest way is to edit the PDB files in a text editor) and 
superpose the fragments. If the whole proteins need to be superposed too, then 
superpose the proteins to their respective fragments. This is especially useful 
when the program does not give you options on residue ranges (for example, COOT 
SSM superpose).

Zhijie


From: WENHE ZHONG 
Sent: Wednesday, October 24, 2012 6:59 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] CCP4superpose_only superpose interesting residues


Dear members,

I have one difficult task on hand and would like to ask for your advice. 

I want to superpose two enzyme structures just based on several residues (e.g. 
5 residues) which we are interested in. But these two structures do not have 
similarity for overall structures. In this case, I can not really use CCP4 
Superpose to do it. May I ask do you have any idea managing to do it? Thank you.

King regards,
Wenhe

Re: [ccp4bb] PNAS on fraud

[Zhijie Li]

Hi Phillippe,

If looking only at the figs. 3A,B,C in the PNAS paper alone, yes, I would 
agree with you that the proposed correlation is quite weak. Without the help 
of the trend lines, I would probably conclude that there is no correlation 
between the IF and number of retractions by a simple look. Of course the R 
squares are quite telling on the quality of the fitting already. On the 
other hand, the same authors had done similar analysis on a smaller pool of 
samples (17 journals) in an earlier study: 
http://iai.asm.org/content/79/10/3855.full, figure 1. It seems that when 
including way less journals, the trend stood out quite nicely - leading the 
authors to say a strong correlation in the earlier publication. I am not 
sure if the earlier clearer trend was a result of cherry picking, as the 
choice of journals looks quite normal – like a standard pool of journals 
one particular lab would consider to publish papers on.


It would also be interesting for us on the CCP4BB to try picking only the 
journals that we would consider to publish structures on, and plot the RI:IF 
graph to see what would happen. Compared to other fields of biology, frauds 
in crystallography is probably easier to detect, thus we need worry less 
about the false negatives: the low impact papers that were fraudulent or 
erroneous, but nobody cared to spend their effort battling.( I think when 
taking consideration of this, drawing conclusions from figs. 3A,B,C  would 
be even more dangerous.)


I would also like to bring two more issues for discussion:

One, in the 2011 IAI paper's fig. 1, the authors plotted Retraction Index, 
which is the total # of Retractions multiplied by 1000 then divided by total 
number of publication, whereas in the 2012 PNAS paper figures 3A,B,C, the 
plots simply used number of retractions to plot against the IFs. I wonder 
what they will look like if the figures 3A,B,C were plotted as RI vs IF – 
considering that many low or moderately-low IF journals publish huge numbers 
of papers.


The second issue is, in the PNAS figures 3A,B,C, at the lower left corner, 
although the dots have a dense looking, the viewers have to realize that 
most of them only represent 1 to 3 retractions. Ten of such points contain 
the same number of retractions that one point at the upper halves of the 
panels A and B contains. Maybe simple bar graphs for numbers of retractions 
in each IF bin would provide more help. Also, the fact that the averaged IFs 
landed at ~8 and ~12 for the fraud and errors cases (fig 3D) suggests that 
the absolute number of retractions occurred in high IF journals is quite 
significant, especially considering that there are way fewer journals with 
IF10 than the ones with IF10 in the 200-300 journals. So in my view, maybe 
trying to fit a straight line to the distribution is overly idealistic, some 
sort of partition does exist.


Zhijie


--
From: DUMAS Philippe (UDS) p.du...@ibmc-cnrs.unistra.fr
Sent: Friday, October 19, 2012 6:15 AM
To: Zhijie Li zhijie...@utoronto.ca
Subject: Re: [ccp4bb] PNAS on fraud



Le Jeudi 18 Octobre 2012 22:52 CEST, Zhijie Li zhijie...@utoronto.ca a 
écrit:


Thank you for this funny (and yet significant) comment.
But I do not see clearly whether you agree with me or with the PNAS 
paper

For me, this conclusion in the PNAS paper is just ridiculous.
Philippe D


On curve fitting:

http://twitpic.com/8jd081


--
From: DUMAS Philippe (UDS) p.du...@ibmc-cnrs.unistra.fr
Sent: Thursday, October 18, 2012 1:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] PNAS on fraud


 Le Jeudi 18 Octobre 2012 19:16 CEST, Bernhard Rupp (Hofkristallrat 
 a.D.)

 hofkristall...@gmail.com a écrit:

 I had a look to this PNAS paper by Fang et al.
 I am a bit surprised by their interpretation of their Fig. 3: they 
 claim
 that here exists a highly signficant correlation between Impact factor 
 and
 number of retractations. Personnaly,  I would have concluded to a 
 complete

 lack of correlation...
 Should I retract this judgment?
 Philippe Dumas

Re: [ccp4bb] PNAS on fraud

[Zhijie Li]

On curve fitting:

http://twitpic.com/8jd081


--
From: DUMAS Philippe (UDS) p.du...@ibmc-cnrs.unistra.fr
Sent: Thursday, October 18, 2012 1:52 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] PNAS on fraud



Le Jeudi 18 Octobre 2012 19:16 CEST, Bernhard Rupp (Hofkristallrat a.D.) 
hofkristall...@gmail.com a écrit:


I had a look to this PNAS paper by Fang et al.
I am a bit surprised by their interpretation of their Fig. 3: they claim 
that here exists a highly signficant correlation between Impact factor and 
number of retractations. Personnaly,  I would have concluded to a complete 
lack of correlation...

Should I retract this judgment?
Philippe Dumas

Re: [ccp4bb] sarkosyl

[Zhijie Li]

Hi,

We used it in some lysis/refolding buffers.

Sigma marked this biodegradable detergent as Highly toxic by inhalation - 
when it's used in toothpastes and shampoos if I am not mistaken.  when I 
called, Sigma said that since someone published a paper saying that when they 
sprayed it into a rat's nose the rat died, they had no choice but to put a 
skull sign on the MSDS.

Zhijie




From: Rashmi Panigrahi 
Sent: Thursday, September 20, 2012 5:47 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] sarkosyl


Hi All,
I was wondering if anyone has used N-lauryl sarcosine in the lysis buffer for 
purifying protein and checked if the protein was functional or structurally okay
Please let me know 
kind regards
-- 
rashmi

Re: [ccp4bb] sarkosyl

[Zhijie Li]

Hi Rashmi,

I can't say that we use it regularly. There are only one or two protocols in 
our lab that need it in the buffers. I didn't do the actual work so I am not 
sure if there was any problem caused by it regarding the protein purification 
or functionality. But those protocols are quite established for the specific 
proteins. I suppose that supports the idea that it at the minimum does not 
always cause problems.

Zhijie


From: Rashmi Panigrahi 
Sent: Thursday, September 20, 2012 8:29 AM
To: Zhijie Li 
Cc: CCP4BB@jiscmail.ac.uk 
Subject: Re: [ccp4bb] sarkosyl


Thanks Zhijie,

So do you mean to say that your lab regularly uses it in Lysis and refolding 
buffers, and the protein/s purified are structurally and functionally fine and 
that there is no aggregation caused due to sarkosyl??

kind regards
Rashmi

On Thu, Sep 20, 2012 at 6:23 PM, Zhijie Li zhijie...@utoronto.ca wrote:

  Hi,

  We used it in some lysis/refolding buffers.

  Sigma marked this biodegradable detergent as Highly toxic by inhalation - 
when it's used in toothpastes and shampoos if I am not mistaken.  when I 
called, Sigma said that since someone published a paper saying that when they 
sprayed it into a rat's nose the rat died, they had no choice but to put a 
skull sign on the MSDS.

  Zhijie




  From: Rashmi Panigrahi 
  Sent: Thursday, September 20, 2012 5:47 AM
  To: CCP4BB@JISCMAIL.AC.UK 
  Subject: [ccp4bb] sarkosyl


  Hi All,
  I was wondering if anyone has used N-lauryl sarcosine in the lysis buffer for 
purifying protein and checked if the protein was functional or structurally okay
  Please let me know 
  kind regards
  -- 
  rashmi




-- 
rashmi

Re: [ccp4bb] off topic: pdf to word conversion

[Zhijie Li]

If you have acrobat (instead of acrobat reader), then you can save PDF to RTF 
file which is fully supported by word. This is probably the easiest way to get 
the maximum out of a PDF file. If this doesn't work to your satisfaction, then 
you can try saving the PDF to TIFF images, then OCR.



From: Rex Palmer 
Sent: Saturday, September 01, 2012 6:48 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] off topic: pdf to word conversion


Dear CCP4BB
Does anyone know how to convert a .pdf file into a meaningful Word file.
Any suggestions will be greatly appreciated. The pdf file has numerous figures 
and tables.

Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.homestead.com

Re: [ccp4bb] off topic: pdf to word conversion

[Zhijie Li]

P.S.
If the images are important and you want to extract them as independent image 
files, then you can try saving the PDF as html. Acrobat will generate an image 
directory that contains the image files. I am using acrobat 8 and there are two 
options, saving as html 3.2 and saving as html 4 with CSS, both worked when I 
tested with a PDF file.
HTH,
Zhijie


From: Rex Palmer 
Sent: Saturday, September 01, 2012 6:48 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] off topic: pdf to word conversion


Dear CCP4BB
Does anyone know how to convert a .pdf file into a meaningful Word file.
Any suggestions will be greatly appreciated. The pdf file has numerous figures 
and tables.

Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.homestead.com

Re: [ccp4bb] Question on stereo monitor: LG D2342P-PN

[Zhijie Li]

Hi Scott, 

The surface doesn't reflect a sharp-edged image so I guess that means it is not 
glossy. But I am not quite sure if it is anti-glare - it does reflect the 
person sitting before it. 

Regards,
Zhijie



From: Scott Thomas Walsh 
Sent: Thursday, August 09, 2012 2:48 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Question on stereo monitor: LG D2342P-PN


Hi Zhijie, 


Is the LG D2342P-PN screen glossy or anti-glare surface?  The Zalman ZM-M240W I 
have is
glossy.


Thank you.


Scott



Scott T. R. Walsh, Ph.D.
Assistant Professor
University of Maryland
IBBR/CBMG
Rm 3127E CARB-2
9600 Gudelsky Drive
Rockville, MD 20850
email: swals...@umd.edu
phone: (240) 314-6478
fax: (240) 314-6255


On Aug 9, 2012, at 2:26 PM, Zhijie Li wrote:


  Hello,

  We have received our LG D2342P-PN monitor yesterday and did a little test. 
  Simply put, it works perfectly for stereo display on COOT and Chimera. We 
  didn't test pymol, as the 0.99 version doesn't have support for the Zalman 
  mode. But the educational version of the new pymol seems to have the Zalman 
  mode.

  Here I list some observations:
  1. We had a Zalman ZM-M215W monitor shortly before this one. The two models 
  are quite comparable to each other.
  2. Both the Zalman and the LG D2342P emit circular polarized light. So 
  simple linear polarization films won't be able to separate the light from 
  the odd and even lines. But the 3D stereo cinemas are of the same type. My 
  3D stereo glasses from a cinema is about 45 degrees counter-clockwise off 
  the LG glasses' axes, but the left and right images are not switched. So it 
  can work OK with the LG monitors, with only a little ghost image, and if I 
  tilt my head to the left then it is as clean as the LG glasses. On the other 
  hand, except for the two pairs of glasses came with the monitor, I haven't 
  figured out where to buy extra glasses yet.
  3. Both Zalman and LG D2342P have wide horizontal viewing angles, but 
  limited vertical viewing angle (maybe 20-40 degrees) for the stereo display 
  (2D display is not limited). This is probably due to that there is a 
  distance between the polarizer layer and the pixel layer so that if viewed 
  from an angle above or below, some light from one row of pixels would have 
  to travel through the line of polarizer one row above or below and then the 
  polarization is mixed - just my guess.
  4. Both Zalman and LG D2342P can work on any computer with a VGA or DVI 
  output and have nothing to do with the operating systems. No special driver 
  is needed. We tested them on our desktops and laptops, in Linux, real and VM 
  Windows. They worked just fine in all cases.

  Regards,

  Zhijie

  --
  From: William G. Scott wgsc...@ucsc.edu
  Sent: Tuesday, July 24, 2012 10:52 AM
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: Re: [ccp4bb] Question on stereo monitor: LG D2342P-PN


Dear Zhijie:



I saw this for under $300 at Amazon's Showroom and wondered the same 

thing.  I can verify that the LG TV will work with coot, etc., as long as 

you turn OFF all stereographic processing and allow the software to do the 

work.  There is an extremely detailed review of this at 


http://www.amazon.com/LG-D2342P-PN-23-Inch-Widescreen-Passive/dp/B004WK3R4U/ref=cm_cr-mr-title
 

by Bill Costa (he gives his email address at the end).  He seems to 

suggest that the 3D glasses for the LG TV are not compatible with this, 

which left me scratching my head.



Having said that, I'll bet it will work fine.  If you can get it from Best 

Buy for under $300 and return it within 30 days for a full refund, it 

might be worth it.



Either way, let us know.



Bill









William G. Scott

Professor

Department of Chemistry and Biochemistry

and The Center for the Molecular Biology of RNA

228 Sinsheimer Laboratories

University of California at Santa Cruz

Santa Cruz, California 95064

USA







On Jul 23, 2012, at 11:42 AM, Zhijie Li wrote:



  Dear CCP4BBers:



  Sorry to bring up this highly repeated topic again. After some internet 

  research, we are about to make the commitment to buy an LG D2342P-PN for 

  setting up a stereo system. I would like to have a final confirmation 

  from someone with real life experience that this model does work with 

  COOT and Chimera the same way as the Zalman monitors. Thanks in advance.



  Zhijie 

[ccp4bb] Question on stereo monitor: LG D2342P-PN

[Zhijie Li]

Dear CCP4BBers:

Sorry to bring up this highly repeated topic again. After some internet 
research, we are about to make the commitment to buy an LG D2342P-PN for 
setting up a stereo system. I would like to have a final confirmation from 
someone with real life experience that this model does work with COOT and 
Chimera the same way as the Zalman monitors. Thanks in advance.

Zhijie

Re: [ccp4bb] harvesting in cold room

[Zhijie Li]

RE: [ccp4bb] harvesting in cold roomDensity of LN2=807g/L, that's roughly 
807/28*22.4=645.6L gas at RT, 1 atm. At 4C the volume will be less: 
645.6/298*277=600L. An empty 2x2x2m cold room=8000L, containing 8000x21%=1680L 
oxygen. 
We normally won't let the whole liter of LN2 to evaporate when freezing 
crystals. But if someone leaves a liter of LN2 in a small cold room and forgets 
to take it out before it all evaporates, then it can become a potential hazard 
to next user. We have to consider that some labs also use cold room as storage 
place, that might also greatly reduce the volume of air inside.
I guess the case could be even worth with CO2, as CO2 gas is denser. If someone 
has a large bag of dry ice subliming in a closed space, then it can fill the 
space from bottom.
So, lighting a candle when work in cold room? I am not sure if the fire 
department will like it.



From: Green, Todd 
Sent: Friday, July 13, 2012 5:51 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] harvesting in cold room


I've noticed that pretty much every time there is an autofill of the dewars in 
the hutches of SSRL or APS, the oxygen sensors go off. but that is a small 
space and many liters of liquid nitrogen. I've frozen routinely in a small cold 
room with a liter or 2 of liquid nitrogen with no issue.


-Original Message-
From: CCP4 bulletin board on behalf of Jacob Keller
Sent: Fri 7/13/2012 4:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] harvesting in cold room

How frequently do the sensors go off?

JPK

On Fri, Jul 13, 2012 at 4:37 PM, David Schuller dj...@cornell.edu wrote:

  On 07/13/12 17:29, Jacob Keller wrote:

 You probably already know this, but nitrogen is not at all
 poisonous--about 78% of the air is nitrogen. I guess you were probably
 worried about asphyxiation?


 We have oxygen sensors in our X-ray hutches for precisely that reason.

 --
 ===
 All Things Serve the Beam
 ===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu




--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


This email was scanned with Mcafee's Anti-Virus appliance, but this
is no guarantee that no virus exists. You are asked to make sure you
have virus protection and that it is up to date.

Re: [ccp4bb] offtopic: packing gel filtration columns

[Zhijie Li]

Hi,

Yes, it can be done by yourself. I repacked our 26/60 column a few years ago 
with homemade apparatus. The column has been working for us since then. 
Basically the loading apparatus was a tubing connecting the top of the 
column and bottom of a reservoir (a flask with an opening near the bottom) 
that contained 30%-50% suspended beads. The length of the tubing was about 
2m, and I left the reservoir on the highest shelf I could find in our lab, 
so that the gravity of the buffer in the column and tubing will drive the 
flow in a constant even manner. It took overnight to get the column nearly 
filled.
The gel filtration columns need to be tightly packed with no discontinuity. 
As I remember, the GE gel filtration packing manual said: push the top 
filter 2mm further down, after the column has been fully compressed by 
flowing buffer at 1cm/min flow rate.
The beads need to be gently suspended in the beginning, but constant 
stirring is not necessary. The flow of the buffer will carry beads into to 
column (because my reservoir has an opening at the bottom). The difficult 
part is at the end, when the column is filled to the top. The beads will be 
compressed quite a lot when you start pumping buffer, then you need to fill 
in more beads. As I remember, I removed the upper filter on the inlet 
connector so that I could load beads through the upper inlet tubing with a 
10mL or 30mL syringe, which generated enough pressure while loading the 
beads - so that I didn't have to take the top off again and again.


There is a manual from GE on how to pack superdex columns. You should be 
able to find it online. The Superdex beads used in different sized columns 
from GE have different diameters. The bigger the column is, the larger the 
beads are. And the larger the beads are, the easier the packing will be. 
16/60 should still be fairly easy to pack. I checked the theoretical plate 
number of our 26/60 after repacking, it was 11000, not as high as the 13000 
stated in the 26/60 new column manual, but it was good enough for us.


Zhijie

--
From: Peter Hsu hsuu...@u.washington.edu
Sent: Thursday, July 12, 2012 9:51 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] offtopic: packing gel filtration columns


Hi all,

Sorry for the slew of offtopic posts, but does anyone here have any 
experience repacking the large 120mL Superdex75/200 columns? Any 
advice/tips on doing it? I've got an older column that's gotten clogged 
while washing w/NaOH (can't go over 0.1mL/min w/o getting overpressure 
alarm), and not sure the bossman would be thrilled w/buying another 
column.


Thanks for any ideas.

Peter 

Re: [ccp4bb] Where to get a thermo-cycling controlled chamber

[Zhijie Li]

Hi Jun,

In this following article an metallic adapter was used to mount a 192 well 
sitting drop plate on a conventional thermocycler.
http://journals.iucr.org/d/issues/2006/05/00/av5047/av5047bdy.html. 
However I guess there would be significant evaporation and condensation if you 
use vapor diffusion setup and cycle the temperatures.

On the other hand, if you do microbatch under oil, then you should be able to 
easily setup the experiment on a 96-tube PCR plate and mount it on a PCR 
machine. I guess the temperature can be more precisely controlled this way with 
no special equipment needed. Especially, the evaporation and condensation 
during thermo cycles would probably be less problematic.

Zhijie



From: Jun Ma 
Sent: Tuesday, June 12, 2012 11:15 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Where to get a thermo-cycling controlled chamber


Hi everybody,

My experiment need me to try a thermo-cycling method for protein 
crystallization. So I need a kind of temperature chamber, which can changes 
temperature quickly, precisely and steadily. Furthermore, it should be 
relatively quiet when running, because I'm afraid vibration will affect the 
crystallization of my protein. I just need a small one, which can control 
temperature from 0 C to 50 C. 
I'm looking for this for some time, and still don't know where to get it. If 
anyone can provide information about where to purchase, or how to solve the 
thermo-cycling control if there's no commercial merchandise, I will appreciate 
that very much.

Any other suggestions are welcome.

Jun Ma

2012-06-12

Re: [ccp4bb] protein precipitate in all PEG kit

[Zhijie Li]

Hi,

The idea used by the Qiagen pre-screen might be relevant to your case:
http://www.qiagen.com/products/protein/crystallization/screeningproteincrystallizationconditions/prescreenassay.aspx
http://www.qiagen.com/literature/handbooks/literature.aspx?id=239

Zhijie



From: fengjuanlv12 
Sent: Monday, June 11, 2012 3:17 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] protein precipitate in all PEG kit


hi all, 
I got a protein with good condition without precipitating in the purification 
process.And the protein is stalbe at 4oC, but when i try crystallization 
screens, it precipating in all the kit(with PEG), in the kit with high 
concentration of salt ,the situation was better(but still no crystal).
any suggestion?
thanks!

fjlv
2012-06-11



fengjuanlv12

Re: [ccp4bb] protein precipitate in all PEG kit

[Zhijie Li]

Hi Fengjuan,

It has been proposed that for a specific protein there are hard precipitants 
and soft precipitants. Hard ones precipitates the the protein effectively, 
while soft ones may help stabilizing the protein. By playing with the two types 
of precipitants, one may get better crystallization results.
Please read here:
http://journals.iucr.org/d/issues/1999/08/00/gr0873/gr0873bdy.html

Zhijie


From: fengjuanlv12 
Sent: Monday, June 11, 2012 3:17 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] protein precipitate in all PEG kit


hi all, 
I got a protein with good condition without precipitating in the purification 
process.And the protein is stalbe at 4oC, but when i try crystallization 
screens, it precipating in all the kit(with PEG), in the kit with high 
concentration of salt ,the situation was better(but still no crystal).
any suggestion?
thanks!

fjlv
2012-06-11



fengjuanlv12

Re: [ccp4bb] Death of Rmerge

[Zhijie Li]

I am little curious about the anisotropically truncated data for 3RKO:

   Percent Possible(All)96.0
Mean I Over Sigma(Observed) 0.8

In the supplementary table of the nature paper it was made clear that this 
3.16-3.0A, I/sigmaI=0.8 and Rmerge=1.216 shell was the outer shell of the 
anisotropically truncated data. The authors did also report the 
isotropically truncated resolution to be 3.2A with I/sigmaI=1.3 and 
Rmerge=73%.


The authors also stated in the main text that

the best native data set was anisotropically scaled and truncated to 3.4 Å, 
3.0 Å and 3.0 Å resolution, where the F/σ ratio drops to ~2.6–2.8 along 
the a*, b* and c* axes, respectively (scaling 2, Supplementary Table 1)


My question is, is the I/sigmaI=0.8 a consequence of many reflections with 
nearly 0 I/sigmaI being included in the calculation? Then what does the 96% 
completeness mean? Does it mean that 96% completeness in the spherical shell 
of 3.16-3.0A was achieved, by including a great number of I=0 reflections?



Zhijie



--
From: Edward A. Berry ber...@upstate.edu
Sent: Thursday, May 31, 2012 2:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Death of Rmerge


Yes! I want a copy of this program RESCUT.

REMARK 200  R SYM FOR SHELL(I) : 1.21700
I noticed structure 3RKO reported Rmerge in the last shell greater
than 1, suggesting the police who were defending R-merge were fighting
a losing battle. And this provides a lot of ammunition to those
they are fighting.

Jacob Keller wrote:

Dear Crystallographers,

in case you have not heard, it would appear that the Rmerge statistic
has died as of the publication of  PMID: 22628654. Ding Dong...?

JPK

--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] SUMO(ULP-1) protease

[Zhijie Li]

Hi Gloria,

I asked for a little baker's yeast from a lab in our department and PCRed 
both the SMT3 domain and the SUMOase gene from its genomic DNA, then cloned 
them to E coli vectors. The good thing about yeast is that most of its genes 
are single exon so one do not really need to find cDNA for most of its 
proteins.
My SUMOase construct was based on the structure pdb 1EUV. It included a 
N-term His6 followed by a bamHI site and the SUMOase 421-601. The sumo 
protease construct can be produced from BL21 at maybe 10-20mg/L and behaved 
quite well. It worked for me when I did cleavage test. However I didn't use 
it extensively due to some problem specific to my protein domain.



SMT3

ETHINLKVSDGSSEIFFKIK
KTTPLRRLMEAFAKRQGKEM
DSLRFLYDGIRIQADQTPED
LDMEDNDIIEAHREQIG




His6-SUMO protease

MHHGSKKLVPELNEKD
DDQVQKALASRENTQLMNRD
NIEITVRDFKTLAPRRWLND
TIIEFFMKYIEKSTPNTVAF
NSFFYTNLSERGYQGVRRWM
KRKKTQIDKLDKIFTPINLN
QSHWALGIIDLKKKTIGYVD
SLSNGPNAMSFAILTDLQKY
VMEESKHTIGEDFDLIHLDC
PQQPNGYDCGIYVCMNTLYG
SADAPLDFDYKDAIRMRRFI
AHLILTDALK


Zhijie

--
From: Gloria Borgstahl gborgst...@gmail.com
Sent: Thursday, May 24, 2012 3:41 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] SUMO(ULP-1) protease


My fellow crystallographers,
We are thinking the SUMO/His vectors would be nice to have in the lab
aresenal... but.  The stumbling block is that the protease needed for
cleavage is very expensive at crystallography scale.  SUMO(ULP-1)
protease costs ~$700/mg fusion protein.   It would not be a problem
for labs using micrograms of protein, but is prohibitive at our level
of protein purification.
Has anyone found a way around this?  Your pal, Gloria 

Re: [ccp4bb] how to ignore spot overlap in imosflm?

[Zhijie Li]

Hi Xinghua,

The total intensity of each reflection needs to be accurately quantitated in 
order to calculate the structure factors. Not only the dots need to be well 
separated in the 3D reciprocal space, but also a small area around the dots are 
often needed to calculate the background for subtraction. That is why when two 
dots are getting too close, the programs will reject both dots. The first thing 
you need to do is to inspect the images reported with large number of overlaps 
to see if the dots are really overlapping or just close to each other. If the 
dots are barely touching or just too close to each other, you can manipulate 
the SEPERATION parameter to force the program to take the closely spaced spots. 
But keep in mind that you may get less accurate integration by doing so. If 
many spots are really touching each other, normally we won't force the programs 
to use them. Then the proper remedy is to move the detector farther and collect 
the dataset again (also, try to optimize your freezing to get the mosaicity as 
low as possible).

For how to play with the mosflm parameters, please read here: 
http://www.mrc-lmb.cam.ac.uk/harry/cgi-bin/keyword2.cgi?SEPARATION. What you 
need is probably CLOSE.

The hazard of high percentage of overlaps:
If the overlaps are only scattered in a whole dataset, it is OK, even if they 
make up 5-10% or even 20% of the whole dataset. It will only give you a lower 
completeness, which is not too detrimental to the structure solution. However, 
if large, continuous regions in the dataset are missing, that will cause you to 
have poorly defined regions in the calculated map, often seen as featureless 
stripes or layers in the map. Unfortunately, when you have closely spaced 
reflections, the latter is often the case. The proper solution is to collect 
the data at a greater detector distance to resolve the spots (after taking the 
test images, both imosflm and HKL2000 can simulate the collection run to help 
you to decide what distance you need). In cases that you have a long unit cell 
(200A), the first thing you need to do is to align the long edge of the Unix 
cell with the rotational axis of the pin. In the difficult cases, you probably 
even need to shoot multiple crystals and combine the datasets to get enough 
completeness.

Zhijie



From: Xinghua Qin 
Sent: Sunday, May 13, 2012 10:22 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] how to ignore spot overlap in imosflm?


Dear CCP4ers,

We collected a diffraction dataset with high percentage of spot overlaps, It 
would be so kind to tell me how to ignore spot overlap in imosflm and explain 
the hazard of high percentage of spot overlaps. 
Thanks in advance.

Best wishes

Xinghua Qin
--
Xinghua Qin
State Key Laboratory of Plant Physiology and biochemistry 
College of Biological Sciences
China Agricultural University
No.2, Yuan Ming Yuan West Road
Haidian District, Beijing, China 100193
Tel: +86-10-62732672
E-mail: xinghua...@126.com

Re: [ccp4bb] Anisotropic diffraction

[Zhijie Li]

Hi Chen,

I see your point now. Yes, I agree that the 80:20 method (or 75:25 as stated 
in the paper, http://www.mail-archive.com/ccp4bb@dl.ac.uk/msg01063.html) is 
a very useful technique. The fact that it does not take much effort or lead 
to uncertainties makes it very worth trying.


Here I add my two cents: when performing such optimizations, try both 
seeding and no-seeding if quantity of protein permits. If protein quantity 
is limited and the crystals are reluctant to appear under the original 
condition, then seeding is always a good idea.


Zhijie




--
From: chen c chenc...@gmail.com
Sent: Sunday, April 29, 2012 6:09 PM
To: Zhijie Li zhijie...@utoronto.ca
Subject: Re: [ccp4bb] Anisotropic diffraction


I accept your advice. In fact, this is the first time I am involved in
anisotropic issue. And I learned a lot from all the above discussion.

However, the 80:20 optimization method(an example of long-tail
theory) rather than surface mutation is what I want to emphasis in my
last email. As illustrated in the attached pdf, it defenitely deserve
a try. One more thing to mention is that this very 80:20 method can be
very versatile and  useful in optimising macromolecular crystals in
case of issues more than anistropic problem.

best regards
chen



2012/4/30 Zhijie Li zhijie...@utoronto.ca:

Hi Chen,

It is a reality that a usable protein dataset could take years of hard 
work
to obtain. Compared to problems such as twining, bad diffraction 
patterns,

excessive mosaicity, low resolution, weak, noisy anomalous signal, etc.,
anisotropic diffraction should probably be regarded most benign. There is
nothing wrong with publishing a properly treated anisotropic dataset. 
Then

why risking another year trying to find a better mutant? There is no
guarantee that the mutants would work better, or even work.

Unless the crystal is naturally isotropic, such as that it is in a cubic
space group, the diffraction of protein crystals will probably always be
more or less anisotropic. This only reflects the fact that the crystal
packing is indeed anisotropic, consequently the movements of the Unit 
cell

is anisotropic. It does not mean that there would be defects in the
resulting structure. For instance, a 3A isotropic dataset, and a 2.5-2A
anisotropic dataset, which one is going to give a better description of 
the

protein?

Zhijie


--
From: chen c chenc...@gmail.com
Sent: Sunday, April 29, 2012 5:03 AM
To: Zhijie Li zhijie...@utoronto.ca

Subject: Re: [ccp4bb] Anisotropic diffraction


If we might be able to eliminate the anisotropic diffraction issue,
which might mean and reflect defect in structure. Why not just
restrain our thought to solve this question by data processing, etc?

chen

2012/4/28 Zhijie Li zhijie...@utoronto.ca:


Hi Cheng,

This paper looks quite irrelevant to Theresa's question.

Zhijie



--
From: chen c chenc...@gmail.com
Sent: Friday, April 27, 2012 10:28 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Anisotropic diffraction



Birtley and Curry used a novel optimization method, in their paper
Crystallization of foot-and-mouth disease virus 3C protease: surface
mutagenesis and a novel crystal-optimization strategy, which might be
inspiring for you.



在 2012年4月28日 上午3:21，David Schuller dj...@cornell.edu 写道：



Anisotropic truncation should have no effect on the space group
symmetry.



On 04/27/12 15:18, Theresa Hsu wrote:




Dear crystallographers

A very basic question, for anisotropic diffraction, does data
truncation
with ellipsoidal method change the symmetry? For example, if
untruncated
data is space group P6, will truncated data index as P622 or P2?

Thank you.

Theresa






--
===
All Things Serve the Beam
===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu






--
Cheng Chen, Ph.D. Candidate
Laboratory of Structural Biology
Life Science Building,Tsinghua University
Beijing 100084
China
Tel:+86-10-62772291
Fax:+86-10-62773145
E-mail:che...@xtal.tsinghua.edu.cn

北京市海淀区清华大学生命科学馆201-212室
邮编：100084








--
Cheng Chen, Ph.D. Candidate
Laboratory of Structural Biology
Life Science Building,Tsinghua University
Beijing 100084
China
Tel:+86-10-62772291
Fax:+86-10-62773145
E-mail:che...@xtal.tsinghua.edu.cn

北京市海淀区清华大学生命科学馆201-212室
邮编：100084







--
Cheng Chen, Ph.D. Candidate
Laboratory of Structural Biology
Life Science Building,Tsinghua University
Beijing 100084
China
Tel:+86-10-62772291
Fax:+86-10-62773145
E-mail:che...@xtal.tsinghua.edu.cn

北京市海淀区清华大学生命科学馆201-212室
邮编：100084

Re: [ccp4bb] Anisotropic diffraction

[Zhijie Li]

Hi,

My first thought was same with David: the truncation won't change the 
crystal's space group. The symmetry of the crystal is reflected by the 
symmetry of the amplitudes of many many reflections across all resolutions. 
Ellipsoidal truncation itself only removes some very weak reflections from 
the outer shells. The remaining reflections will still have a good number of 
reflections carrying the symmetry of the crystal.


However a second thought on the anisotropic scaling and B-factor correction 
led me to this scenario: suppose we have a crystal that's really P6, but we 
have cowardly indexed it to a lower space group P2, with the 2-fold axis, b, 
coinciding the real 6-fold axis. By losing the a=c restrain, the anisotropic 
scaling along H and L now may not be strictly equal (for example, could be 
caused by outliers that would have been identified and filtered out if 
indexed correctly as P6), resulting in the loss of the 6-fold symmetry in 
the scaled dataset. Apparently this is an artifact due to an improper SG 
assignment before the anisotropic scaling and B-factor correction.


Just some crazy thoughts. Please correct me if I am wrong.



BTW, to Theresa: an very informative introduction on ellipsoidal truncation 
and anisotropic scaling can be found here:


http://services.mbi.ucla.edu/anisoscale/



--
From: Theresa Hsu theresah...@live.com
Sent: Friday, April 27, 2012 3:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Anisotropic diffraction


Dear crystallographers

A very basic question, for anisotropic diffraction, does data truncation 
with ellipsoidal method change the symmetry? For example, if untruncated 
data is space group P6, will truncated data index as P622 or P2?


Thank you.

Theresa