[ccp4bb] Modeling sugars with Coot and Phenix

[Emilia C. Arturo (Emily)]

Hello all,


I have several questions regarding sugar modeling. These are questions for
both Coot (0.9.4-pre/CCPEM) and Phenix (1.18.2-3874) usage (on a Mac) for
modeling as correctly as possible several N-linked glycosylation (i.e. not
ligand) sugars within a protein, and about the approach as it pertains to
higher resolution X-ray maps (better than 2A) or to lower resolution EM
maps (~3-4A). I am new to the world of glycoproteins and cryoEM, not new to
model building or X-ray crystallography, and would welcome any advice or
insight.


For relatively low resolution maps, where sugar moieties cannot be placed
unambiguously, in theory (say Agirre  (2017) and Emsley and Crispin (2018))
pyranose placement within the map should be quite highly restrained by
ideal geometry for the specific sugar expected from prior knowledge. In
practice, however, it’s not entirely clear how to maintain these restraints
on pyranose geometry and conformation during manual building in Coot, or
how to apply any special restraints in Phenix. The Glyco module of Coot
does indeed place fine looking Asn-NAGs after a few iterations of manual
placing and refining to account for the poor resolution of the acetyl
group, just as discussed by Emsley and Crispin (2018). However, the
validation report that is obtained from the OneDep system at the rcsb web
server for the resulting structure shows many outliers in bonds and angles
within the NAG or between the NAG and protein Asn residue. In Coot I have
experimented with various restraints (in the R/RC menu), including
decreasing the Refinement weight/weight matrix to zero, or some small
non-zero number, thinking this places less weight on the map signal and
more on the ideal values within Coot’s libraries. I have tried a variety of
refinement restraint combinations in Coot to address individual bonds and
angles outliers within the sugar, but the rcsb validation report still
finds departures from ideal geometry. Meanwhile, in Phenix I don’t do
anything specific for the refinement of glycosylation sugars (should I?)
other than apply the appropriate restraints for any low resolution EM map;
are there specific restraints files I ought to be using within Phenix real
space refine jobs for N-linked sugars?


Then I noticed that loads of existing PDB entries containing N-linked
glycans contain similar outliers in pyranose geometry. So this prompted me
to wonder whether we’re not doing things right, that in practice we’re not
correctly following what’s advised in theory, or whether this is just the
state of model building currently. Could someone more experienced than am I
on sugar building please comment here on how exactly to get the sugars
better built? e.g. what buttons to press and why, in Phenix and/or in Coot.
Or is this a tolerable situation where sugar geometries differ from ideal
even at low resolution? Is it perhaps a matter of different targets being
used for PDB validation by the rcsb versus by Coot?


It turns out that regarding higher resolution maps I am also unsure of
exactly how to apply restraints within Coot. For example, using PDB ID 5VAA
and its map with a reported resolution of 1.55A, I centered on Asn297 and
its associated glycosylation sugars. I experimented with refinement
strategies (i.e. the settings of R/RC), thinking that the best approach
would be little restraint on ideal geometry and higher weight on the map
signal. But no matter what combination of restraints I used, I was unable
to keep the model locally from distorting; i.e. the deposited model versus
map looks great, but within Coot I find a hundred ways to destroy it. I
must be doing something wrong.


Any advice or references you’d recommend would be greatly appreciated.


Thanks.

-- 
"Study as if you were going to live forever; live as if you were going to
die tomorrow." - Maria Mitchell

"I'm not afraid of storms for I'm learning to sail my ship."  - Louisa May
Alcott



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[ccp4bb] powdery residue on pucks?

[Emilia C. Arturo (Emily)]

Hi All,

We have been noticing lately that our crystal pucks return from different
beamlines coated with the same powdery material. For the record, we
typically undo our pucks, clean the assemblies and dry out the
pucks within a day of receiving the shipping dewar, but notice the same
thing regardless of how long between receiving the dewar and disassembling
the pucks. Have any of you experienced this sort of thing, or have
suggestions of what might be the cause and/or the powdery material?

Regards,
Emily.

-- 
"Study as if you were going to live forever; live as if you were going to
die tomorrow." - Maria Mitchell

"I'm not afraid of storms for I'm learning to sail my ship."  - Louisa May
Alcott



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Re: [ccp4bb] Searching similar local structural conformations

[Emilia C. Arturo (Emily)]

Lei,

The DeGrado lab developed a tool that would do just what you seek to do and
could function as a PyMOL plugin. I played with it a few years ago after I
found an interesting cation-pi sandwhich in my structure. I e-mailed then
with the author, hoping to link the tool to the entire current PDB
inventory, but then had to move on to another project, so am not certain
how updated it is now.  Have a look at the tool and documentation here:
http://degradolab.org/suns/ It is reported in 2014 by Gonzalez et al, here:
https://www.ncbi.nlm.nih.gov/pubmed/25079944

Regards,
Emily.

On Wed, Jan 8, 2020 at 7:57 AM Zheng, Lei  wrote:

> Dear CCP4ers,
>
>
>
> We identify a potential ion binding site formed by four residues in a
> structure. I want to search if any other structures have a similar local
> residual geometry. Is there any programs to perform such a searching? For
> example, to search in Protein Data Bank using coordinates of the binding
> site residues? I appreciate your suggestions.
>
>
>
> Happy New Year!
>
> Lei
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>


-- 
"Study as if you were going to live forever; live as if you were going to
die tomorrow." - Maria Mitchell

"I'm not afraid of storms for I'm learning to sail my ship."  - Louisa May
Alcott



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[ccp4bb] Research technician position at La Jolla Institute for Immunology (California, USA)

[Emilia C. Arturo (Emily)]

The Ollmann Saphire laboratory at La Jolla Institute for Immunology (LJI)
is seeking a technician to work as a part of our team. We are focused on
infectious diseases and immune response in general, viral pathogens that
cause hemorrhagic fever in particular. The lab has a solid core of
structural biologists, virologists and molecular biologists. We are seeking
a technician to aid primarily in cloning and protein production, though
opportunities abound to learn and contribute to various aspects of our
crystallography and tomography efforts, as well as antibody design,
antibody discovery and cell culture.

LJI is located in beautiful San Deigo, CA. You can find out more about LJI,
its mission, breadth of research and approach to human health, here:
https://www.youtube.com/watch?v=PbV1CjlriC8#action=share

You can find out more about the Ollmann Saphire laboratory here
https://www.lji.org/faculty-research/labs/saphire/#overview. Please contact
Dr. Erica Ollmann Saphire directly at er...@lji.org with leads or inquiries
about this position.

Regards,
Emilia C Arturo
Postdoctoral associate, Ollmann  Saphire laboratory
La Jolla Institute for Immunology, Division of Structural Biology &
Infectious Diseases

-- 
"Study as if you were going to live forever; live as if you were going to
die tomorrow." - Maria Mitchell

"I'm not afraid of storms for I'm learning to sail my ship."  - Louisa May
Alcott



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Re: [ccp4bb] A helix with leucine repeats

[Emilia C. Arturo (Emily)]

Could it be a member of a coiled coil at some point in its lifetime, as a
part of its function in regulating position or activity of this receptor?
Does the helix have sequence similarity to other coiled coils? see
https://www.uniprot.org/help/coiled for a primer on the topic.

Looks fun!

Emily.



On Sun, Mar 4, 2018 at 5:01 PM, Cheng Zhang  wrote:

> Hi Michael,
>
> Thanks for the information.
>
> It is amphipathic. It follows a transmembrane helical domain of a cell
> surface receptor and adopts an orientation parallel to the membrane. So it
> may be associated with the membrane. I am just wondering if such
> leucine-repeat motif is special among all amphipathic, membrane-associated
> helical structures. In other words, any other possible functional roles
> than just membrane association?
>
> Best,
>
> Cheng
>
>
> On Sat, Mar 3, 2018 at 10:23 PM, R. Michael Garavito  > wrote:
>
>> Dear Cheng,
>>
>> Chris and Ruud have provided you with the typical interpretation of such
>> a motif, but you have forgotten to give the CCP4 community the context of
>> this leucine-repeat helix.  Is it amphipathic?  Does the protein also have
>> transmembrane helices (as suggested by the figure provided) which would
>> provide the membrane anchoring?
>>
>> Look up structurally similar membrane proteins (not necessarily
>> homologous by sequence), such as proteins which have a monotonic-like
>> membrane interaction, but that also have a transmembrane helix (monoamine
>> oxidase or some mammalian cyt P450s).  If your protein is monotopic, look
>> at that class of membrane proteins (squalene cyclase, fatty acid hydrolase,
>> cyclooxygenase, etc.).  One structurally undescribed motif is a “reentrant
>> membrane helix.”  In other words, look at context before assigning
>> function.
>>
>> Good luck and hope this helps,
>>
>> Michael
>>
>> **
>>
>> *R. Michael Garavito, Ph.D.*
>>
>> *Professor of Biochemistry & Molecular Biology*
>>
>> *513 Biochemistry Bldg.   *
>>
>> *Michigan State University  *
>>
>> *East Lansing, MI 48824-1319*
>>
>> *Office:*  *(517) 355-9724 <(517)%20355-9724> Lab:  (517) 353-9125
>> <(517)%20353-9125>*
>>
>> *FAX:  (517) 353-9334 <(517)%20353-9334>Email:
>> rmgarav...@gmail.com *
>>
>> **
>>
>>
>>
>> On Mar 3, 2018, at 3:51 PM, Cheng Zhang > > wrote:
>>
>> Hi all,
>>
>> We recently got a structure of a transmembrane protein. There is a helix
>> that is parallel to the membrane. The function of this helix is not known
>> and we are trying to make some hypothesis. A unique feature is that there
>> are repeated leucine residues on this helix facing the lipids. I am
>> wondering if anyone has seen a similar pattern and could suggest possible
>> function, e.g. membrane anchoring?
>>
>> Thanks!
>>
>> Best,
>>
>> Cheng
>>
>> 
>>
>>
>> --
>> -
>> Cheng Zhang
>>
>>
>>
>
>
> --
> -
> Cheng Zhang
>



-- 
"Study as if you were going to live forever; live as if you were going to
die tomorrow." - Maria Mitchell

"I'm not afraid of storms for I'm learning to sail my ship."  - Louisa May
Alcott

Re: [ccp4bb] cannot read h5 data file

[Emilia C. Arturo (Emily)]

Shijun,

I have processed .h5 format data successfully that was collected at NSLS II
AMX (this beamline has a Eiger 9M, not 16M, incidentally, but I don't think
that this matters). I used XDSGUI without converting to .cbf format. To do
so, and without knowing what the error messages are that you are receiving
as output, it is important that you include the following two lines, at
minimum, in your XDS.INP file: DETECTOR= EIGER and include the correct
'LIB=' line; for instructions on the latter, see the XDSWiki page here:
https://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Eiger#Script_for_generating_XDS.INP_from_master.h5
, and get the XDS Neggia plugin here:
https://www.dectris.com/company/news/newsroom/news-details/process-eiger-data-with-xds-fast
. nb You will need to register to download the plugin, and *do* read the
README file.  :-)

Wishing you well,

Emily.

On Wed, Dec 20, 2017 at 3:50 AM, 张士军 <21620150150...@stu.xmu.edu.cn> wrote:

> Dear all
>
>I just collected some data with Eiger16M whose file format is *.h5. But
> I cannot read it with HKL2000, because I don't have the Eiger16M detector
> license now, while, I have *.cbf detector license. So I transfered the *.h5
> to *.cbf files, but the problem is  XDSGUI still cannot read them,and
> HKL2000 can read it, can search peak, but cannot index it with warning me
> ''no enough peaks to index the data'',when I check the peaks are much
> enough to index, whearas dials can read  it, but the cell parameters are
> not the same with before. Anyone can tell me what's wrong with it? Thanks a
> lot !!!
>
> Best Regards
>
> Shijun
>



-- 
"Study as if you were going to live forever; live as if you were going to
die tomorrow." - Maria Mitchell

"I'm not afraid of storms for I'm learning to sail my ship."  - Louisa May
Alcott

Re: [ccp4bb] Differences in a homodimer protein

[Emilia C. Arturo (Emily)]

I assume that somehow the two subunits are distinguishable despite their
forming a "homodimer." Otherwise I don't know how you would know that it is
always the same subunit that contains the water molecule. If they are truly
distinguishable, then, the first thing that's come to mind is the
possibility that this asymmetric dimer structure is demonstrating a symptom
of operating via a half-of-the-sites reactivity mechanism in solution. This
would be interesting. I don't know by what mechanism it would be an
artifact of the crystallization or structure solution.

Regards,
Emily.

On Tue, Nov 28, 2017 at 2:04 PM, Denis Rousseau <
denis.rouss...@einstein.yu.edu> wrote:

> Hi BB members
>
> I have a homodimeric protein, which contains metal centers. In several
> different derivatives I find a water molecule on a metal center in one
> subunit which is absent on the other.  It is always the same subunit that
> contains the water molecule. The resulution is ~2.4 A. Is this an artifact
> or a functional difference? If it is truly homodimeric I would expect
> differences to be random. The space group is P212121.
>
> Thanks for the advice.
>
> Denis Rousseau
> --
>
>


-- 
"Study as if you were going to live forever; live as if you were going to
die tomorrow." - Maria Mitchell

"I'm not afraid of storms for I'm learning to sail my ship."  - Louisa May
Alcott

Re: [ccp4bb] doubt regarding MR search model

[Emilia C. Arturo (Emily)]

Hello Randy,

I'm chiming in about the last sentence in your reply:


> Finally, I would suspect that getting a significantly lower LLG for two
> copies of a dimer means that the dimer in your structure is slightly
> different from the dimer in the model.
>

Will you please be more specific about what you consider "significantly
lower" (or higher) for LLG scores? And how does this difference translate
to differences among TFZ scores? I have wondered this especially when a
Phaser MR result from use of a smaller part (i.e. one domain, 64% of the
sequence) of a structure differs by a few units relative to a result from
use of a larger part (i.e. two domains, 90% of the sequence) of the
protein; the difference in my case was a top TFZ score of 42.5 using the
one-domain search model, but a lower TFZ score of 36.9 for the two-domain
model.

Even more interesting to me is when the TFZ scores vary by quite a bit more
between two phaser runs that used two different, but 100% sequence
identical, one-domain search models; in this case the top TFZ scores were
18.7 versus 42.5, for the two models that differed only in the coordinate
position of ~20 out of 290 residues (a lid for the enzyme).

Regards,

Emily.


Best wishes,
>
> Randy Read
>
> -
> Randy J. Read
> Department of Haematology, University of Cambridge
> Cambridge Institute for Medical ResearchTel: +44 1223 336500
> Wellcome Trust/MRC Building Fax: +44 1223 336827
> Hills Road
> E-mail: rj...@cam.ac.uk
> Cambridge CB2 0XY, U.K.
> www-structmed.cimr.cam.ac.uk
>
> > On 18 Sep 2017, at 16:24, Andrew Leslie 
> wrote:
> >
> > Regarding the warning message "The top solution from a TF rescoring did
> not pack”, I get this on all the PHASER jobs that I have run recently, but
> looking through the PHASER log file I cannot see any evidence for packing
> failure.
> >
> > It may be that the failure is buried in an obscure place in the very
> long log file, but if I search for “pack” then I get the output summarised
> below, all of which, apart from the ADVISORY, suggests to me that there is
> no problem at all with the packing.
> >
> > Perhaps someone on the PHASER team can cast light on this.
> >
> > Andrew
> >
> >
> > Extracts from PHASER log file:
> >
> >
> > TRANSLATION FUNCTIONS
> > -
> >
> >Target Function: FAST LETF1
> >Translation Packing Function applied: top peak will pack
> >Translation Packing Cutoff: 50%
> >Sampling:  1.82 Angstroms
> >
> >
> > ===
> > 
> > PACKING FUNCTION
> > 
> >
> >There are 4 solutions to pack
> >Packing analysis
> >0%100%
> >|=| DONE
> >
> > 
> >Packing Table
> >-
> >Solutions accepted if pairwise clashes less than 10 % of trace atoms
> >#in  #out Clash-% Symm TF-SET  ROT TFpk#TFTFZ
> SpaceGroup
> >1Top1  0.741  --   1 11325.94 26.03  P 1
> >22 1.058  --   1 21293.29 24.34  P 1
> >33 0.952  --   1 31245.70 21.88  P 1
> >44 0.847  --   1 41211.49 20.11  P 1
> >
> >4 accepted of 4 solutions
> >   4 pack of 4 accepted solutions
> >
> > ==
> > ---
> > TRANSLATION PACKING
> > ---
> >
> >Translation Packing Cutoff: 50%
> >All solutions have been packed
> >
> > ==
> >Packing Fast Search Translations...
> >   9871 peaks
> >   500 peaks over 1049.74 checked for packing
> >   Translation peak 1 first to be kept
> >   Done
> >
> > 
> >New Top Packing Fast Translation Function FSS = 2181.37 (TFZ=46.3) at
> Trial #1
> > 
> >
> >Top Peaks Without Clustering
> >Select peaks over 67.5% of top (i.e. 0.675*(top-mean)+mean)
> >There was 1 site over 67.5% of top
> >1 peak selected
> >The sites over 67.5% are:
> ># Frac X Frac Y Frac ZFSS   Z-score
> >1  0.719  0.122  0.890 2181.4 46.35
> >
> >Top 1 translations before clustering will be rescored
> >Calculating Likelihood for TF SET #1 of 1 TRIAL #1 of 1
> >0% 100%
> >|==| DONE
> >
> >Packing LLG Translations: pass 1 of 11...
> >   1 peaks
> >   No peaks over 1878.46 - no packing check
> >Packing LLG Translations: pass 2 of 11...
> >   1 peaks
> >   1 peaks over 1878.46 checked for packing
> >   Translation peak 1 first to be kept
> >   Done
> >   Exit: found a peak that packs
> >
> > -==
> >
> > *** AND THEN ***
> > 
> --
> > Advisory: The top solution from a TF rescoring did not pack
> > 
> 

Re: [ccp4bb] just out of totally idle curiosity ...

[Emilia C. Arturo (Emily)]

I had previously considered to relocate, at least temporarily. But now a
part of me wants to stay and fight for what we (as scientists) have managed
to achieve.

On Nov 9, 2016 12:45 AM, "Tom Peat"  wrote:

> I don't know about Europe, but it is very tight Down Under...
>
>
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> William G. Scott
> Sent: Wednesday, 9 November 2016 4:38 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] just out of totally idle curiosity ...
>
> What’s the job situation in Europe looking like for refugee scientists
> these days?
>
>
>
> William G. Scott
> Director, Program in Biochemistry and Molecular Biology Professor,
> Department of Chemistry and Biochemistry and The Center for the Molecular
> Biology of RNA University of California at Santa Cruz Santa Cruz,
> California 95064 USA
>
> http://scottlab.ucsc.edu
>
>

Re: [ccp4bb] PyMOL v. Coot map 'level'

[Emilia C. Arturo (Emily)]

Thomas,


 I tried to figure out the PyMOL vs. Coot normalization discrepancy a while
 ago. As far as I remember, PyMOL normalizes on the raw data array, while
 Coot normalizes across the unit cell. So if the data doesn't exactly cover
 the cell, the results might be different.


I posted the same question to the Coot mailing list (the thread can be
found here: https://goo.gl/YjVtTu) , and got the following reply from Paul
Emsley; I highlight the questions that I think you could best answer, with
'***':

[ ...]
I suspect that the issue is related to different answers to the rmsd of
what?

In Coot, we use all the grid points in the asymmetric unit - other programs
make a selection of grid points around the protein (and therefore have less
solvent).

More solvent means lower rmsd. If one then contours in n-rmsd levels, then
absolute level used in Coot will be lower - and thus seem to be noisier
(perhaps).  I suppose that if you want comparable levels from the same
map/mtz file then you should use absolute levels, not rmsd. ***What does
PyMOL's 1.0 mean in electrons/A^3?***

Regards,

Paul.

Regards,
Emily.


 On 01 Jun 2015, at 11:37, Emilia C. Arturo (Emily) ec...@drexel.edu
 wrote:
 One cannot understand what is going on without knowing how this map
  was calculated.  Maps calculated by the Electron Density Server have
  density in units of electron/A^3 if I recall, or at least its best
  effort to do so.
 
  This is what I was looking for! (i.e. what the units are) Thanks. :-)
  Yes, I'd downloaded the 2mFo-DFc map from the EDS, and got the same Coot
 v. PyMOL discrepancy whether or not I turned off the PyMOL map
 normalization feature.
 
 If you load the same map into Pymol and ask it to normalize the
  density values you should set your contour level to Coot's rmsd level.
   If you don't normalize you should use Coot's e/A^3 level.  It is
  quite possible that they could differ by a factor of two.
 
  This was exactly the case. The map e/A^3 level (not the rmsd level) in
 Coot matched very well, visually, the map 'level' in PyMOL; they were
 roughly off by a factor of 2.
 
  I did end up also generating a 2mFo-DFc map using phenix, which fetched
 the structure factors of the model in which I was interested. The result
 was the same (i.e. PyMOL 'level' = Coot e/A^3 level ~ = 1/2 Coot's rmsd
 level) whether I used the CCP4 map downloaded from the EDS, or generated
 from the structure factors with phenix.
 
  Thanks All.
 
  Emily.
 
 
 
  Dale Tronrud
 
  On 5/29/2015 1:15 PM, Emilia C. Arturo (Emily) wrote:
   Hello. I am struggling with an old question--old because I've found
   several discussions and wiki bits on this topic, e.g. on the PyMOL
   mailing list
   (http://sourceforge.net/p/pymol/mailman/message/26496806/ and
   http://www.pymolwiki.org/index.php/Display_CCP4_Maps), but the
   suggestions about how to fix the problem are not working for me,
   and I cannot figure out why. Perhaps someone here can help:
  
   I'd like to display (for beauty's sake) a selection of a model with
   the map about this selection. I've fetched the model from the PDB,
   downloaded its 2mFo-DFc CCP4 map, loaded both the map and model
   into both PyMOL (student version) and Coot (0.8.2-pre EL (revision
   5592)), and decided that I would use PyMOL to make the figure. I
   notice, though, that the map 'level' in PyMOL is not equivalent to
   the rmsd level in Coot, even when I set normalization off in PyMOL.
   I expected that a 1.0 rmsd level in Coot would look identical to a
   1.0 level in PyMOL, but it does not; rather, a 1.0 rmsd level in
   Coot looks more like a 0.5 level in PyMOL. Does anyone have insight
   they could share about the difference between how Coot and PyMOL
   loads maps? Maybe the PyMOL 'level' is not a rmsd? is there some
   other normalization factor in PyMOL that I should set? Or, perhaps
   there is a mailing list post out there that I've missed, to which
   you could point me. :-)
  
   Alternatively, does anyone have instructions on how to use Coot to
   do what I'm trying to do in PyMOL? In PyMOL I displayed the mesh of
   the 2Fo-Fc map, contoured at 1.0 about a 3-residue-long
   'selection' like so: isomesh map, My_2Fo-Fc.map, 1.0, selection,
   carve=2.0, and after hiding everything but the selection, I have a
   nice picture ... but with a map at a level I cannot interpret in
   PyMOL relative to Coot :-/
  
   Regards, Emily.
  -BEGIN PGP SIGNATURE-
  Version: GnuPG v2.0.22 (MingW32)
 
  iEYEARECAAYFAlVo1L4ACgkQU5C0gGfAG10YkwCfROYPVXBK/pDS4z/zi5MNY1D+
  nHIAnjOFiAkb6JbuIGWRWkBFDG5Xgc2K
  =hrPT
  -END PGP SIGNATURE-
 

 --
 Thomas Holder
 PyMOL Principal Developer
 Schrödinger, Inc.

Re: [ccp4bb] PyMOL v. Coot map 'level'

[Emilia C. Arturo (Emily)]

One cannot understand what is going on without knowing how this map
 was calculated.  Maps calculated by the Electron Density Server have
 density in units of electron/A^3 if I recall, or at least its best
 effort to do so.


This is what I was looking for! (i.e. what the units are) Thanks. :-)
Yes, I'd downloaded the 2mFo-DFc map from the EDS, and got the same Coot v.
PyMOL discrepancy whether or not I turned off the PyMOL map normalization
feature.


If you load the same map into Pymol and ask it to normalize the
 density values you should set your contour level to Coot's rmsd level.
  If you don't normalize you should use Coot's e/A^3 level.  It is
 quite possible that they could differ by a factor of two.


This was exactly the case. The map e/A^3 level (not the rmsd level) in Coot
matched very well, visually, the map 'level' in PyMOL; they were roughly
off by a factor of 2.

I did end up also generating a 2mFo-DFc map using phenix, which fetched the
structure factors of the model in which I was interested. The result was
the same (i.e. PyMOL 'level' = Coot e/A^3 level ~ = 1/2 Coot's rmsd level)
whether I used the CCP4 map downloaded from the EDS, or generated from the
structure factors with phenix.

Thanks All.

Emily.



 Dale Tronrud

 On 5/29/2015 1:15 PM, Emilia C. Arturo (Emily) wrote:
  Hello. I am struggling with an old question--old because I've found
  several discussions and wiki bits on this topic, e.g. on the PyMOL
  mailing list
  (http://sourceforge.net/p/pymol/mailman/message/26496806/ and
  http://www.pymolwiki.org/index.php/Display_CCP4_Maps), but the
  suggestions about how to fix the problem are not working for me,
  and I cannot figure out why. Perhaps someone here can help:
 
  I'd like to display (for beauty's sake) a selection of a model with
  the map about this selection. I've fetched the model from the PDB,
  downloaded its 2mFo-DFc CCP4 map, loaded both the map and model
  into both PyMOL (student version) and Coot (0.8.2-pre EL (revision
  5592)), and decided that I would use PyMOL to make the figure. I
  notice, though, that the map 'level' in PyMOL is not equivalent to
  the rmsd level in Coot, even when I set normalization off in PyMOL.
  I expected that a 1.0 rmsd level in Coot would look identical to a
  1.0 level in PyMOL, but it does not; rather, a 1.0 rmsd level in
  Coot looks more like a 0.5 level in PyMOL. Does anyone have insight
  they could share about the difference between how Coot and PyMOL
  loads maps? Maybe the PyMOL 'level' is not a rmsd? is there some
  other normalization factor in PyMOL that I should set? Or, perhaps
  there is a mailing list post out there that I've missed, to which
  you could point me. :-)
 
  Alternatively, does anyone have instructions on how to use Coot to
  do what I'm trying to do in PyMOL? In PyMOL I displayed the mesh of
  the 2Fo-Fc map, contoured at 1.0 about a 3-residue-long
  'selection' like so: isomesh map, My_2Fo-Fc.map, 1.0, selection,
  carve=2.0, and after hiding everything but the selection, I have a
  nice picture ... but with a map at a level I cannot interpret in
  PyMOL relative to Coot :-/
 
  Regards, Emily.
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 Version: GnuPG v2.0.22 (MingW32)

 iEYEARECAAYFAlVo1L4ACgkQU5C0gGfAG10YkwCfROYPVXBK/pDS4z/zi5MNY1D+
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[ccp4bb] PyMOL v. Coot map 'level'

[Emilia C. Arturo (Emily)]

Hello.
I am struggling with an old question--old because I've found several
discussions and wiki bits on this topic, e.g. on the PyMOL mailing list (
http://sourceforge.net/p/pymol/mailman/message/26496806/ and
http://www.pymolwiki.org/index.php/Display_CCP4_Maps), but the suggestions
about how to fix the problem are not working for me, and I cannot figure
out why. Perhaps someone here can help:

I'd like to display (for beauty's sake) a selection of a model with the map
about this selection. I've fetched the model from the PDB, downloaded its
2mFo-DFc CCP4 map, loaded both the map and model into both PyMOL (student
version) and Coot (0.8.2-pre EL (revision 5592)), and decided that I would
use PyMOL to make the figure. I notice, though, that the map 'level' in
PyMOL is not equivalent to the rmsd level in Coot, even when I set
normalization off in PyMOL. I expected that a 1.0 rmsd level in Coot would
look identical to a 1.0 level in PyMOL, but it does not; rather, a 1.0 rmsd
level in Coot looks more like a 0.5 level in PyMOL. Does anyone have
insight they could share about the difference between how Coot and PyMOL
loads maps? Maybe the PyMOL 'level' is not a rmsd? is there some other
normalization factor in PyMOL that I should set? Or, perhaps there is a
mailing list post out there that I've missed, to which you could point me.
:-)

Alternatively, does anyone have instructions on how to use Coot to do what
I'm trying to do in PyMOL? In PyMOL I displayed the mesh of the 2Fo-Fc map,
contoured at 1.0 about a 3-residue-long 'selection' like so: isomesh map,
My_2Fo-Fc.map, 1.0, selection, carve=2.0, and after hiding everything but
the selection, I have a nice picture ... but with a map at a level I cannot
interpret in PyMOL relative to Coot :-/

Regards,
Emily.

Re: [ccp4bb] Role of protein dimerization

[Emilia C. Arturo (Emily)]

 Dear All,



 I have a question that is a little bit related to the previous discussion
about crystallisation of a minority fraction monomers. I wonder if there is
a review of some sort (or anything in principle) that would discuss role of
dimerization (or more broadly oligomerization) in proteins in general. It’s
clear that dimerization can be used for regulation (only one species is
active), but for other dimers this on/off mechanism is not important. I’m
just curious if someone did any comparisons…


A review article from our lab, published in 2012, I think, provides an
eloquent description of the role of oligomerization in controlling protein
function. The review also introduces a newly observed allosteric
mechanism--the morpheein model--but does so by giving a historical
perspective on the evolution of allosteric models in general. The paper,
Dynamic dissociating homo-oligomers and the control of protein
function is by Selwood and Jaffe in Arch Biochem Biophys. 2012 Mar
15;519(2):131-43, found here http://www.ncbi.nlm.nih.gov/pubmed/22182754

Regards,
Emily Arturo
Jaffe lab, Fox Chase Cancer Center
Philadelphia, PA






 Thank you!



 Natalia

Re: [ccp4bb] Offtopic: Software to closely visualize interacting partnets in protein complex

[Emilia C. Arturo (Emily)]

As I'm sure you've also found, it's not simple to find one [easily
accessible] program that examines and reports every type of
interaction that might be of interest to you. So I'm sending in a
reference to another web-based tool that I've found complementary to
PISA and the others mentioned here. In particular, it calculates
features like the gap volume, number of segments, and the secondary
structure-type that dominates between two chains. The tool is called
2P2I Inspector, and can be found here
http://2p2idb.cnrs-mrs.fr/2p2i_inspector.html along with other tools
and databases that might also interest you.

Wishing you well,
Emily Arturo

Ph.D. program in Biochemistry, Drexel Univ College of Medicine
Jaffe lab, Fox Chase Cancer Center
Philadelphia, PA

On Wed, Mar 11, 2015 at 1:24 PM, Tim tim.schu...@rub.de wrote:
 Hi,
 Molprobity is also a nice software to do this kind of analysis - if you use
 it as implemented in phenix you also get good visualization in coot.
 I also asked the pymol community to create an implementation for pymol, but
 I did not follow if somebody took that up.

 Also this 'protein interactions calculation' server is very neat:
 http://pic.mbu.iisc.ernet.in/

 /Tim


 On 10/03/15 11:25, Debasish Kumar Ghosh wrote:

 Dear All,

 Apologies for this little off-topic inquiry. I want to closely visualize
 the interacting residues in an multimeric protein complex to understand the
 nature of interactions. Is there any good software to give this information
 with good clarity.
 Any suggestion is highly appreciated.

 Thanks,
 Best !!!

 Debasish Kumar Ghosh

 CSIR- Junior Research Fellow (PhD Scholar)
 C/o: Dr. Akash Ranjan
 Computational and Functional Genomics Group
 Centre for DNA Fingerprinting and Diagnostics
 Hyderabad, INDIA

 Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
 Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
 Lab URL:
 http://www.cdfd.org.in/labpages/computational_functional_genomics.html

Re: [ccp4bb] protein quantification with carbohydrates

[Emilia C. Arturo (Emily)]

Tianyu,

My suggestion is to determine the molar absorption coefficient empirically
using the Edelhoch method (
http://www.rpgroup.caltech.edu/courses/PBL/bootcamp_June07/pdf/articles/Edelhoch1967.pdf).
This involves measuring the absorbance at 280nm of your native and your
unfolded protein (a nice protocol and explanation here:
http://mail.ypu.edu.tw/~wnhuang/apparatus/ps0301.pdf), and then measuring
your protein concentration using this absorption coefficient in
Beer-Lambert's law.

Emily.

On Tue, Mar 10, 2015 at 5:49 PM, Gmail adams.wan...@gmail.com wrote:

   Dear all,



  I have a (heavily and heterogeneously) glycosylated protein that has
 very low OD280 absorption. I wondered whether the glycans will have any
 effect on the accuracy of BCA or Bradford assay. Amino acid analysis
 doesn’t seem to be any better because carbohydrates will interfere with
 sample analysis and quantification. Any suggestion about other methods that
 we could try to measure the concentration of this protein? Much appreciated!



 Best,

 Tianyu

Re: [ccp4bb] Skin on drops

[Emilia C. Arturo (Emily)]

Artem,


 In addition to other answers, one of the more esoteric (but surprisingly
 effective) ways to destroy the protein 'skin' on drops is to add a tiny bit
 of Trypsin solution.


Will you go a bit further and say what exactly you mean by a tiny bit of
Trypsin solution? :-)  What is the composition of your successful trypsin
solution? In what volume do you add it? Do you add it right on top of a
sitting or hanging drop? Was your situation one in which the skin and
crystals were fused? Was there a rationale for why you used trypsin as
opposed to other peptidases (I imagine it's what you had on hand ...)?

Emily.



 Crystals generally tend not to be affected, but (at least in my
 experience) the protein skin dissolves within a couple of hours or less. In
 at least one case, this was the only way to get rid of skin, which
 otherwise tugged on crystals and resulted in considerably worse diffraction.

 Good luck,

 Artem

 P.S. needless to say, if your skin is made of something else then
 trypsinolysis won't work :)

 - Cosmic Cats approve of this message

 On Wed, Feb 25, 2015 at 2:34 AM, Ulrike Demmer 
 ulrike.dem...@biophys.mpg.de wrote:

 Dear crystallographers,

 I am trying to crystallize a soluble protein which tends to form
 aggregates. The crystallization condition is 20% PEG 3350 + 0,2 M
 Na-Formate. During the crstallization process a thick skin is formed on top
 of the sitting-drops. As well the crystals are buried in precipitate.
 Before I start harvesting I try to remove the skin but still it is hardly
 possible to get any crystals out of these drops.

 Any suggestions how to avoid the formation of skin on crystallization
 drops ?

 Cheers,

 Ulrike

[ccp4bb] clarification Re: Density fit analysis in Coot, and FEM

[Emilia C. Arturo (Emily)]

Thank you for the multiple kind off-list responses I received regarding how
to interpret map colors in Coot. I'm very grateful for the references, but
it seems that I did not state my issue clearly :-) What I was referring to
was the tool that Coot has under Validate  Density Fit analysis. The tool
outputs graphs like the ones I'm pasting below. Both sets of graphs were
generated from the same pdb file, but the very-red bar graphs were
calculated using the map coefficients phenix had generated along with this
model, while the mostly green bar graphs pasted separately below, showing
bars for the same stretch of residues in each chain, were generated using
the feature enhanced map that phenix generated from this same model.[image:
Inline image 1][image: Inline image 2] How do I interpret the fact that the
FEM and 2Fo-Fc maps give such different fits for the same model using this
type of analysis? ...or are the fits really that different (and maybe green
versus red is not as big as the visual cue would have me assume)?

Emily.

On Thu, Feb 19, 2015 at 11:00 AM, Emilia C. Arturo (Emily) ec...@drexel.edu
 wrote:

 Hello all.
 I'd like to understand what it is I'm looking at when I use Coot's density
 fit analysis tool. I recognize that there was a post related to this
 topic on the Coot bb a while ago --the discussion was on how to interpret
 the red-ness or green-ness of the density fit plot (
 https://www.mail-archive.com/coot@jiscmail.ac.uk/msg02995.html)
 https://www.mail-archive.com/coot@jiscmail.ac.uk/msg02995.html)--but --but
 it doesn't seem the issue was resolved then (i.e. what does 'red' really
 mean? is it ...bad?). Now I have more to ask that involves my using
 phenix-generated FEMs to build in Coot.

 So what I've done is the following: I adjust my model in Coot, using a
 phenix-generated FEM as the map for fitting, then refine with phenix, and
 using the refined pdb and reflections file, I use phenix to generate a new
 FEM. Then I repeat. At some point I learned about Coot's density fit
 analysis tool and took a look at how my model fits. If the map that is
 selected in the sidebar of Coot is a FEM, then the density fit analysis
 plot looks mostly green everywhere - fine. If, however, I select as my map
 in the Coot sidebar the 2Fo-Fc that phenix had generated along with the
 latest refined model--the one I'm examining with the density fit analysis
 tool--then Coot's density fit analysis plot looks red (with values ~ 
 0.3), with splashes of orange, barely any green or yellow (with values ~ 
 0.3), almost everywhere.

 So these are my questions: What are the units of the density fit values?
 i.e. What is the calculation that's done? I'm surprised that the
 FEM-dependent density fit graphs look so different (i.e. so green) relative
 to the graphs generated if my map is set to the 2Fo-Fc from the loaded
 model; both maps came from the same model. In fact, I got worried, but then
 I realized that I don't actually understand the red-ness and green-ness.
 I'm quite new to the business of crystallography so any input is welcome
 regarding the use of FEMs and density fit analyses.

 Emily.
 Ph.D. program in Biochemistry, Drexel Univ. College of Medicine
 Jaffe lab, Fox Chase Cancer Center
 Philadelphia, PA

[ccp4bb] Density fit analysis in Coot, and FEM

[Emilia C. Arturo (Emily)]

Hello all.
I'd like to understand what it is I'm looking at when I use Coot's density
fit analysis tool. I recognize that there was a post related to this
topic on the Coot bb a while ago --the discussion was on how to interpret
the red-ness or green-ness of the density fit plot (
https://www.mail-archive.com/coot@jiscmail.ac.uk/msg02995.html)
https://www.mail-archive.com/coot@jiscmail.ac.uk/msg02995.html)--but --but
it doesn't seem the issue was resolved then (i.e. what does 'red' really
mean? is it ...bad?). Now I have more to ask that involves my using
phenix-generated FEMs to build in Coot.

So what I've done is the following: I adjust my model in Coot, using a
phenix-generated FEM as the map for fitting, then refine with phenix, and
using the refined pdb and reflections file, I use phenix to generate a new
FEM. Then I repeat. At some point I learned about Coot's density fit
analysis tool and took a look at how my model fits. If the map that is
selected in the sidebar of Coot is a FEM, then the density fit analysis
plot looks mostly green everywhere - fine. If, however, I select as my map
in the Coot sidebar the 2Fo-Fc that phenix had generated along with the
latest refined model--the one I'm examining with the density fit analysis
tool--then Coot's density fit analysis plot looks red (with values ~ 
0.3), with splashes of orange, barely any green or yellow (with values ~ 
0.3), almost everywhere.

So these are my questions: What are the units of the density fit values?
i.e. What is the calculation that's done? I'm surprised that the
FEM-dependent density fit graphs look so different (i.e. so green) relative
to the graphs generated if my map is set to the 2Fo-Fc from the loaded
model; both maps came from the same model. In fact, I got worried, but then
I realized that I don't actually understand the red-ness and green-ness.
I'm quite new to the business of crystallography so any input is welcome
regarding the use of FEMs and density fit analyses.

Emily.
Ph.D. program in Biochemistry, Drexel Univ. College of Medicine
Jaffe lab, Fox Chase Cancer Center
Philadelphia, PA

Re: [ccp4bb] Crystallization problem

[Emilia C. Arturo (Emily)]

 On Mon, Jan 26, 2015 at 8:22 PM, Monica Mittal monica.mitta...@gmail.com
 wrote:

 Hi everyone,

 I need an advice on some strange thing happening to one of the protein i
 am working on. I used to purify it and set up trays and get some needle
 shaped crystals and trying seeding and other methods to optimise them. But
 recently, it stopped giving crystals even small needles. I am still
 following the same protocol with same buffer stocks.


By 'buffer stocks', do you also mean your stock of precipitating reagent?
It's happened to me that a hit I pursued and optimized vanished when I
opened a new bottle of PEG. It took too long to realize that the old PEG
could have been at a higher concentration than I'd assumed, given its
shelf-age. I increased the PEG % and got back the crystals.

Good luck,
Emily.


And not just once but since last three times it is happening. The purified
 protein in gel filtration is perfectly fine eluting at same position with
 symmetrical distribution. However when i am setting up trays under previous
 conditions, i am not getting the crystals. Instead the drops are quite
 clear. So i increased the concentration of the protein also from 8 to
 11mg/ml, but still the same. Infact i tried adding ligand also but again no
 crystals. So i would be really grateful if anyone can give a valuable
 suggestion regarding this problem !!

 Thanks
 BR
 Monica

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

[Emilia C. Arturo (Emily)]

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 03f1d08ed29c-dmarc-requ...@jiscmail.ac.uk
 *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] Demonstration for 2nd graders?

[Emilia C. Arturo (Emily)]

When my daughter was in Kindergarten, her class took a trip to our
facility, and I showed them some of my crystal trays (What do you see
here? Do you see anything?  Clear drops ..., they effectively said).
Then I showed them through the microscope several crystals, and I was
pleasantly surprised by their awe (oooh! Jewels!). Then I showed them
some loops by-eye and by-microscope. I'd have liked to show them how I
manipulate a crystal with that loop, but I wasn't set up to project it for
everyone to see real-time. I then told them about protein machines that
line up in a particular way to form that jewel they'd just seen. ...I
imagine you cannot bring a good enough microscope into the classroom with
you without some hassle, but I thought I'd share at least that 5-6 year
olds, even, can find protein crystals very fascinating.

Emily.

On Thu, Jan 8, 2015 at 2:02 PM, David Schuller schul...@cornell.edu wrote:

  This one is probably above second grade, but the equipment setup is
 pretty easy

 http://ipl.physics.harvard.edu/wp-uploads/2013/03/15c_s07_5.pdf
 Measuring the wavelength of light with a ruler





 On 01/08/15 13:35, John Lee wrote:

 Hi everyone,

  Slightly off topic here but I got myself volunteered by my 2nd grade son
 to do a show and tell at his class. I have the rock candy experiment ready
 with some background info on what I do.

  Can anyone direct me to some resources or your personal demo's that you
 have done?

  Thanks a bunch

  -John




 --
 ===
 All Things Serve the Beam
 ===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu

Re: [ccp4bb] asymmetric homotrimer in the asu

[Emilia C. Arturo (Emily)]

Hay,


 Indeed, we also incline to think of it as a monomer in solution,


It is in fact possible that different solution conditions favor different
oligomeric assemblies. For example, perhaps your protein, which in one set
of solution conditions prefers the monomer, prefers some other assembly
under the crystallization conditions (we've seen this in our own work).
Perhaps the higher (or lower) salt concentration, the presence (or absence)
of the precipitation reagent, etc. pushes any oligomer equilibrium that
exists for your protein.

Regardless of what our final conclusion would be for this case, we became
 rather generally interested to find other similar cases of *homomeric*
 assemblies related only by non crystallographic translation symmetry (or as
 Engin Qzka pointed out improper NCS is the conventional terminology). So
 to rephrase our question we are interested to learn about additional
 structures of *homomoeric improper ncs assemblies*.


An interesting case of heterologous interfaces engaged is that of the VP40
protein. A recent publication from the Saphire group (PMID 23953110
http://www.ncbi.nlm.nih.gov/pubmed/23953110) may present an instructive
situation, though I don't know the details of the NCS observed. Here an
octameric assembly (PDB 4LDM, ASU=monomer) and a hexameric assembly (PDB
4LDD, ASU=dimer) of the same protein engage different interfaces on the
protomers. In forming the oligomer, the protomers are aligned with
alternating interfaces.

Emily.


 I truly appreciate ANY open-minded or skeptic thought, profound or trivial
 that we get here! They all, definitely those made by Mark Garavito,
 contribute to shaping our mind around this riddle.
 Thanks for commenting on the skepticism, I brought it up as part of the
 discussion but a glitch of my own coffee time haziness might have slipped
 in. Perhaps I should try some o-cha instead .. :)

 cheers,
 Hay


 On Dec 12, 2014, at 3:05 AM, Jeremy Tame wrote:

 Dear Hay

 I suggest that you use analytical ultracentrifugation to determine the
 oligomeric state of the protein in solution.
 Mass spectrometry and light scattering are also useful, but there are so
 many examples of gel filtration proving
 erroneous it has questionable value as an analytical technique. For an
 example of a dimer interface predicted by
 PISA to be real you could look at Yoshida et al, JMB 423, 351 (2012). The
 protein is in fact a monomer in solution.
 PISA is a fantastic tool, but interfaces in crystals do not always reflect
 the solution state. My guess (with the
 information I have) is that your protein is probably a monomer too.

 With regard to Michael Garavito's reply requesting more information, I
 would like to comment that scepticism
 is indeed an important god in the pantheon of science, but that that minor
 deity open-mindedness also deserves the
 occasional nod. 10-fold crystal symmetry is one example, but the list of
 impossible things now become mainstream
 is a long one (continental drift, Earth 100,000 years old, quantum
 mechanicsand so on). Bayes theorem cannot
 help you discover the truth if you have set its prior probability to zero.
 But I haven't my morning o-cha yet either.

 good luck
 Jeremy


 On Dec 11, 2014, at 9:27 PM, Hay Dvir wrote:

 Dear all,



 We have a structure of a rather tightly packed homotrimer protein in the
 ASU with no apparent crystallographic or non-crystallographic rotational
 symmetry between monomers.

 Attempting to establish the biological assembly, we are very interested to
 hear about additional similar cases you might know of.


 Thanks in advance,

 Hay



 ---

 Hay Dvir Ph. D.

 Head Technion Center for Structural Biology

 Technion Haifa 323, Israel

 Tel:  +(972)-77-887-1901

 Fax:  +(972)-77-887-1935

 E-mail hd...@technion.ac.il

 Website http://tcsb.technion.ac.il