Re: [ccp4bb] symmetry possibilities

[Edward A. Berry]

As for your second question, whether refining in a lower symmetry affects the 
R-factors,

On 7/20/22 10:10 AM, Jorge Iulek wrote:

     Related, and a question I mentioned before in this forum: what if a 
genuine 2 axis (say , P222 to P2, or even to P1) (I do not mean this is the 
case here) is ignored such that one would have doubled the number of 
observations but also doubled the number of parameters to be refined (suppose 
to exclude NCS in any case). Would one expect R/Rfree values to be similar in 
both P222 and P2 (or even P1)? How much extra freedom would one have besides 
the twin domain fractions to refine?


The experiment described here:
  https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg46635.html
addresses this, although it wasn't the main point of that email.

The structures 1CHR and 2CHR are derived from the same crystal. Data was 
collected in I4 and 1CHR was published with this space group, and with the low 
symmetry data.  It was later concluded that the true space group was I422. The 
original data was reduced to that space group and the structure solved again. 
In the experiment described in the email referenced above, the model of 2chr 
was refined using (then) current phenix.refine. In parallel the 2CHR structure 
was expanded to a dimer and refined against the original low-symmetry data.

Because the number of reflections was low (even with 10% free) the uncertainty 
in R-free was high, so the refinement was repeated 10 times in each case, with 
different randomly selected free sets, to get the average. In each case the 
refinement was carried out for 50 macrocycles to approach convergence. The R 
and Free-R values were not significantly different.
eab

From that email:
2. The deposited structure was refined in I422 10 times, 50 macrocycles
each, with randomly picked 10% free reflections
 R: 0.1725±0.0013   Rfree: 0.2507±0.0062  Nfree: 908.9±   Nrefl: 9087

3. The structure was expanded to an I4 dimer related by the unused I422
crystallographic operator, matching the dimer of 1chr. This dimer was
refined against the original (I4) data of 1chr, picking free reflections
in symmetry related pairs. This was repeated 10 times with different
random seed for picking reflections.
R: 0.1666±0.0012   **Rfree:0.2523±0.0077   Nfree: 1601.4  Nrefl:16011

4. same as 3 but picking free reflections randomly without regard for
lattice symmetry.
On average 15 free reflections were in pairs, 212 were invariant under
the operator (no sym-mate) and 1374 (86%) were paired with working
reflections.
R: 0.1674±0.0017   **Rfree:0.2523±0.0050  Nfree: 1600.9 Nrefl:16011



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Re: [ccp4bb] [EXTERNAL] [ccp4bb] renaming chains

[Edward A. Berry]

I have noticed renaming chains in the sequence ID's but not in the coordinates:

2FBW_3|Chains C, G[auth P]|Succinate dehydrogenase cytoch

MATTAKEEMARFWEKNTKSSRPLSPHISIYKWSLPMAMSITHRGTGVALSLGVSLFSL

2FBW_4|Chains D, H[auth Q]|Succinate dehydrogenase [ubiqu

GSSKAASLHWTSERAVSALLLGLLPAAYLYPGPAVDYSLAAALTLHGHWGLGQVITDY

2FBW_1|Chains A, E[auth N]|Succinate dehydrogenase [ubiqu

STKVSDSISTQYPVVDHEFDAVVVGAGGAGLRAAFGLSEAGFNTACVTKLFPTRSHTV

2FBW_2|Chains B, F[auth O]|Succinate dehydrogenase [ubiqu

AQTTSRIKKFSIYRWDPDKPGDKPRMQTYEVDLNKCGPMVLDALIKIKNELDST

CHAINS NOPQ (AUTH) are labeled EFGH in the "view FASTA"
The coordinates still have the oddball lettering. Which could be confusing.

Ed



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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Problem with peakmax

[Edward A. Berry]

On 06/10/2021 11:19 AM, James Holton wrote:


I'm afraid the only bash command I know is: "tcsh"


I learned csh by studying "elves"



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Re: [ccp4bb] Ice ring data issue

[Edward A. Berry]

(Obviously this is for culling the bad data after data reduction is complete- 
doesn't do anything to prevent the ice ring from introducing error in the 
scaling process. Not really what you were asking for, and maybe not even useful 
now, given automatic outlier rejection in today's programs)

On 03/05/2021 04:00 PM, Edward A. Berry wrote:

For manually masking out the rings, you can use mtz2various to dump reflections 
in resolution ranges without ice in ascii, cat them all together and use f2mtz 
to read them into a new mtz file. Example attached.

You might also be able to do it using mtzutils to write out mtz files in 
limited resolution ranges, and then to merge them back into one file.

On 03/04/2021 10:39 AM, Alexander Brown wrote:

Hi all,
I'm struggling with a dataset I have which shows very poor data quality around 
3.6A, or exactly where I can see a significant ice ring in the images. I'm 
trying to use mosflm to process the image files, and I have seen a previous 
thread on the message board where it is recommended to turn on three tick boxes 
for ice ring exclusion, but despite this, as I continue through the processing 
sequence and use aimless, it still flags that the data is affected by an ice 
ring at that resolution, which you can also see in the quality/resolution 
graphs.

I have even tried making a mask in the moslfm viewer using the mask tool to 
cover the entire ice ring, but to no avail.

Finally I did have a go at using EVAL which is mentioned in the original post 
about ice rings, but it seems it depends on libgfortran3 packages which have 
now been replaced with libgfortran5 and so I didn't get very far.

Is there a manual way to mask out certain data, or could there be something 
with my data that is causing the automatic ice ring resolution not to be as 
effective?

Thank you!

*Alex Brown*


PhD Student

School of Pharmacy

Biodiscovery Institute (previously Centre for Biomolecular sciences)

University of Nottingham

Nottingham

NG7 2RD


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Re: [ccp4bb] [EXTERNAL] [ccp4bb] Ice ring data issue

[Edward A. Berry]

For manually masking out the rings, you can use mtz2various to dump reflections 
in resolution ranges without ice in ascii, cat them all together and use f2mtz 
to read them into a new mtz file. Example attached.

You might also be able to do it using mtzutils to write out mtz files in 
limited resolution ranges, and then to merge them back into one file.

On 03/04/2021 10:39 AM, Alexander Brown wrote:

Hi all,
I'm struggling with a dataset I have which shows very poor data quality around 
3.6A, or exactly where I can see a significant ice ring in the images. I'm 
trying to use mosflm to process the image files, and I have seen a previous 
thread on the message board where it is recommended to turn on three tick boxes 
for ice ring exclusion, but despite this, as I continue through the processing 
sequence and use aimless, it still flags that the data is affected by an ice 
ring at that resolution, which you can also see in the quality/resolution 
graphs.

I have even tried making a mask in the moslfm viewer using the mask tool to 
cover the entire ice ring, but to no avail.

Finally I did have a go at using EVAL which is mentioned in the original post 
about ice rings, but it seems it depends on libgfortran3 packages which have 
now been replaced with libgfortran5 and so I didn't get very far.

Is there a manual way to mask out certain data, or could there be something 
with my data that is causing the automatic ice ring resolution not to be as 
effective?

Thank you!

*Alex Brown*


PhD Student

School of Pharmacy

Biodiscovery Institute (previously Centre for Biomolecular sciences)

University of Nottingham

Nottingham

NG7 2RD


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icefilter.csh
Description: C-Shell script

Re: [ccp4bb] Pymol Map range

[Edward A. Berry]

On 03/04/2021 10:42 AM, Gerard DVD Kleywegt wrote:

If there is interest I could post a tgz file with -as far as I can determine- 
the latest version of all manuals in HTML format (0.8 MB total).)

Yes, I would like to grab a copy of the manuals.
Ed



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Re: [ccp4bb] Depositing data reduced with HKL2000 - ~half as many reflections in log file statistics

[Edward A. Berry]

This came up on phnix BB recently:

On 10/20/2020 01:30 PM, John Berrisford wrote:

Dear Armando and Pavel

During the deposition process we compare two values

_reflns.number_obs



And
_refine.ls_number_reflns_obs

We expect the number of observed reflections to be higher (or the same) as
the number of reflections used in refinement.
We would expect that these two mmCIF items handle Friedel pairs consistently
- if the number of reflections observed is reported with Friedel pairs
separately then the number of reflections in refinement should also report
Friedel pairs separately.

My assumption here is that there is a mismatch in reporting these values.



On 10/20/2020 02:16 PM, Boaz Shaanan wrote:

Hi Pavel,
When I marked explicitly 'no ano' in the gui (whenever I didn't want to use 
anomalous), this doubling on the output file was gone. As I recall it also 
depends on the labels one chooses to use from the data file. This was on older 
builds though.
Cheers,
Boaz



On 11/21/2020 02:42 PM, Igor Petrik wrote:

This may be a basic question, but a few google searches did not turn up 
anything helpful.

I am trying to deposit a structure, from data reduced with HKL2000 and solved 
and refined with Phenix. The data were scaled with the Anomalous option 
selected.

For depositing, I upload the coordinates, the *_refine_data.mtz file from Phenix, and the *.log file 
from HKL2000 scaling. When I do this, the PDB deposition website complains on the Data Collection 
Statistics page that the "Number of unique reflections measured" (16343) "Has value 
< Total number of reflections refined against (see Refinement page) [23562]"

When I open the original sca file in Phenix, I see:
Number of Miller indices: 31365
Bijvoet pairs: 15030


What is the correct way to reconcile these numbers for the deposition?

Thanks,
- Igor Petrik, PhD

P.S. I collected and processed the data 5+ years ago, and no longer have access 
to HKL2000.

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Re: [ccp4bb] Does MicroED (microcrystal electron diffraction) have phase problem ?

[Edward A. Berry]

For my own info,
Does microED encompass work with 2D crystals, or only micro-3D crystals?

On 08/15/2020 10:40 PM, Jessica Bruhn wrote:

Hi Alex,

Welcome to the field of microED! From a practical standpoint, microED also 
suffers from the phase problem, and somewhat moreso compared to X-ray 
crystallography because anomalous signal is very limited. It is true that a TEM 
microscope operated in imaging mode produces images that contain both phase and 
amplitude information, which you correctly infer means that single particle 
cryoEM does not suffer from the phase problem. This is why a map from cryoEM is 
generally of higher quality than one determined by X-ray crystallography at the 
same resolution, because we don't have to guess at the phases.

In classic microED data collection, we don't actually take advantage of the 
phase information that can be measured using imaging mode. We quickly find our 
crystals in imaging mode and then flip the switch to diffraction mode and 
collect a dataset devoid of phase information. It has been suggested that we 
could use imaging mode to get at the phases of the diffraction patterns, but I 
am not aware of anyone actually doing this. Practically speaking, this would 
add significant additional time to the data collection and we likely would only 
be able to reliably use the phases for the lower resolution range (though, that 
might not be a deal breaker). Thankfully though, molecular replacement and ab 
initio methods for small molecules work pretty well on these datasets. Plus, 
most X-ray structures are solved this way anyways.

There have been efforts to use radiation damage as a means to phase a small peptide bound to zinc 
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313391/ 
).
 And there is some interesting work being done with dynamical scattering as a way to see differences 
in Friedel pairs (see Tim Gruene's post here: 
https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=CCP4BB;b22eae56.2007 
),
 but these methods are not "classic microED" and require completely different data 
processing software that is unfamiliar to most X-ray crystallographers.

For now, we just crank up the cycles in SHELXT/SHELXD or hope for a good 
molecular replacement model.

Best of luck,
Jessica

On Sat, Aug 15, 2020 at 6:03 PM Alex Lee mailto:alexlee198...@gmail.com>> wrote:

Hi, All,

I am new to MicroED (microcrystal electron diffraction). I know that X-ray 
crystallography has phase problem, and I think MicroED has phase problem too 
(it is diffraction of electron instead of x-ray). However, when I read the 
Wikipedia, I could not understand the following description of MicroED: One of 
the main difficulties in X-ray crystallography is determining phases in the 
diffraction pattern. Because of the complexity of X-ray lenses, it is difficult 
to form an image of the crystal being diffracted, and hence phase information 
is lost. Fortunately, electron microscopes can resolve atomic structure in real 
space and the crystallographic structure factor phase information can be 
experimentally determined from an image's Fourier transform. The Fourier 
transform of an atomic resolution image is similar, but different, to a 
diffraction pattern—with reciprocal lattice spots reflecting the symmetry and 
spacing of a crystal.

Does the above description mean that MicroED, or more broadly electron 
crystallogaphy does NOT suffer from phase problem?  How about single particle 
cryo electron microscopy, it should NOT have phase problem, right?

Thanks for any input in it.

Best,
Alex


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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Accessing full list of programs in CCP4I2

[Edward A. Berry]

There seems to be a list of the 7.0 programs (and documentation!) still 
available:
http://www.ccp4.ac.uk/html/FUNCTION.html
I hope this is not going away!
eab

On 07/07/2020 12:09 PM, Tim Gruene wrote:

Dear Kelvin,

the command 'ls $CBIN' lists the majority of CCP4 programs on your
system. This probably even works under Windows 10, since the powershell
acts like a linux/unix shell.

Best regards,
Tim

On Tue, 7 Jul 2020 16:00:18 +
Lau Kelvin  wrote:


Ah I see! Great I can run it that way

And yes, Eleanor, after I realized that list was gone, I was
panicking that there are some random things I used to do with those
programs would no longer be possible. Good to know that the command
line versions are still there in the 7.1 distro.

Best,

Kelvin

--
Kelvin Lau
https://people.epfl.ch/kelvin.lau

Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch
Phone: +41 21 69 30267
If unreachable: +41 21 69 34494


On 07.07.20, 15:51, "CCP4 bulletin board on behalf of Christian Roth"
mailto:CCP4BB@JISCMAIL.AC.UK> on behalf of
christianroth...@gmail.com> wrote:

yes Eleanor is right. command line still works.:-)
fft is also in 7.1 distribution.



On Tue, Jul 7, 2020 at 3:43 PM Eleanor Dodson
mailto:eleanor.dod...@york.ac.uk>> wrote:
Oh Lau - how I miss that list! But if you just run fft online it is
still distributed..wombat:Downloads eleanor$


fft hklin  mapout 

LABIN FP=  and so on..


On Tue, 7 Jul 2020 at 14:22, Christian Roth
mailto:christianroth...@gmail.com>>
wrote: Hi Kelvin, well fft as single program is kind of not longer
supported as is not ccp4i. In i2 internally, as well in communication
with mg or coot, everything is done using map coefficients. Their are
two options: First via i2: Use the unusual map coefficients Task and
choose not to compare maps, but to generate the map coefficients plus
a map (button is in Advanced tab) the standard grid parameters can be
changed, but are actually optimized already. Second just save the map
out of Coot (Export map)

To avoid redundancy, the old fft task was discontinued. i2 works with
coefficients, which generates smaller files and Coot provides all the
functions to generate the map and is its own gui.

Hope that explains a bit why things are how they are now.

Cheers
Christian

On Tue, Jul 7, 2020 at 1:56 PM Lau Kelvin
mailto:kelvin@epfl.ch>> wrote: Hi Christian,

I was in particular looking for the fft program (I couldn’t find that
using the method you described) just to convert an mtz into a .map.
Before in version 7.0 I could just browse all programs, now it seems
like I cannot do that (other than using the filter, and some seem to
be missing)

At the end I just used mtz2map in phenix.

--
Kelvin Lau
https://people.epfl.ch/kelvin.lau

Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch
Phone: +41 21 69 30267
If unreachable: +41 21 69 34494


On 06.07.20, 13:34, "Christian Roth"
mailto:christianroth...@gmail.com>> wrote:

Hi Kelvin,
not quite sure if I understand you correctly,  but if you press the
Task Manager button you get on the right sight a list of topics
(import data, Molecular Replacemnt etc.) each point can be open up
like a file tree to see all programs or pipelines available. You can
search with the search field (Filter) on top for specific program
names. Does that help?

Cheers
Christian

On Mon, Jul 6, 2020 at 1:15 PM Lau Kelvin
mailto:kelvin@epfl.ch>> wrote: Hello,

I am looking for a way to find the list of programs accessible using
the new 7.1 CCP4I2 interface? Is this still possible or do I have to
revert back to version 7.0?

Best regards,

Kelvin

--
Kelvin Lau
https://people.epfl.ch/kelvin.lau

Protein production and structure core facility - PTPSP
EPFL SV PTECH PTPSP
AI 2146 (Bâtiment AI)
Station 19
CH-1015 Lausanne
Switzerland
Email: kelvin@epfl.ch
Phone: +41 21 69 30267
If unreachable: +41 21 69 34494




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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] number of frames to get a full dataset?

[Edward A. Berry]

Now can we get rid of all the superfluous disks in our RAID? Or at least not 
replace them when they fail?

On 06/29/2020 06:24 PM, Andreas Förster wrote:

I like to think that the reflections I carefully measured at high multiplicity are not redundant, which the 
dictionary on my computer defines as "not or no longer needed or useful; superfluous" and the 
American Heritage Dictionary as "exceeding what is necessary or natural; superfluous" and 
"needlessly repetitive; verbose".

Please don't use the term Needless repetitivity in your Table 1.  It sends the 
wrong message.  Multiplicity is good.

All best.


Andreas



On Tue, Jun 30, 2020 at 12:03 AM James Holton mailto:jmhol...@lbl.gov>> wrote:

I have found that the use of "redundancy" vs "multiplicity" correlates very well with the 
speaker's favorite processing software.  The Denzo/HKL program scalepack outputs "redundancy", whereas 
scala/aimless and other more Europe-centric programs output "multiplicity".

At least it is not as bad as "intensity", which is so ambiguous as to be 
almost useless as a word on its own.

-James Holton
MAD Scientist

On 6/24/2020 10:27 AM, Bernhard Rupp wrote:


> Oh, and some of us prefer the word 'multiplicity' ;-0

Hmmm…maybe not. ‘Multiplicity’ in crystallography is context sensitive, and 
not uniquely defined. It can refer to 

 1. the position multiplicity (number of equivalent sites per unit cell, 
aka Wyckoff-Multiplicity), the only (!) cif use of multiplicity
 2. the multiplicity of the reflection, which means the superposition of 
reflections with the same /d/  (mostly powder diffraction) 
 3. the multiplicity of observations, aka redundancy.

While (a) and (b) are clearly defined, (c) is an arbitrary experimental 
number.

How from (a) real space symmetry follows (b) in reciprocal space (including 
the epsilon zones, another ‘multiplicity’) is explained here 

https://scripts.iucr.org/cgi-bin/paper?a14080 

 

and also on page 306 in BMC.

Too much multiplicity might create duplicity… 

Cheers, BR

__ __

Jon Cooper

__ __

On 23 Jun 2020 22:04, "Peat, Tom (Manufacturing, Parkville)" mailto:tom.p...@csiro.au>> wrote:

I would just like to point out that for those of us who have worked too 
many times with P1 or P21 that even 360 degrees will not give you 'super' 
anomalous differences. 

I'm not a minimalist when it comes to data- redundancy is a good thing 
to have. 

cheers, tom 

__ __

Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au  

__ __




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*To:* CCP4BB@JISCMAIL.AC.UK  
mailto:CCP4BB@JISCMAIL.AC.UK>>
*Subject:* Re: [ccp4bb] number of frames to get a full dataset? 



Someone told me there is a cubic space group where you can get away 
with something like 11 degrees of data. It would be interesting if that's 
correct. These minimum ranges for data collection rely on the crystal being 
pre-oriented, which is unheard-of these days, although they can help if someone 
is nagging you to get off the beam line or if your diffraction fades quickly. 
Going for 180 degrees always makes sense for a well-behaved 

Re: [ccp4bb] REMARK 280 in mmcif? (and thoughts about "% w/v")

[Edward A. Berry]

On 01/26/2020 01:02 PM, benjamin bax wrote:





Data items in the PDBX_EXPTL_CRYSTAL_GROW_COMP category record
details about the components of the solutions that were 'mixed'
to produce the crystal.
http://mmcif.wwpdb.org/dictionaries/mmcif_pdbx_v40.dic/Categories/pdbx_exptl_crystal_grow_comp.html 


loop_
_pdbx_exptl_crystal_grow_comp.crystal_id
_pdbx_exptl_crystal_grow_comp.sol_id
_pdbx_exptl_crystal_grow_comp.comp_id
_pdbx_exptl_crystal_grow_comp.comp_name
_pdbx_exptl_crystal_grow_comp.conc
_pdbx_exptl_crystal_grow_comp.conc_range
_pdbx_exptl_crystal_grow_comp.conc_units
4'protein' 1  'protein'   25..  'mg/ml'
4'protein' 2  'Tris HCl'  20..  'millimolar'
4'protein' 3  'NaCl'   0.2   .  'molar'
4'precipitant' 1  'PEG 4000'  12.5   .  'percent_weight_by_volume'
4'precipitant' 2  'MES'0.1   .  'molar'
pdbout copy remarks 280



One argument against PDBX_EXPTL_CRYSTAL_GROW_COM is that it perpetuates (at least in this example) the 
incorrect/deprecated use of "%w/v". This is a measure of the mass concentration of the component, 
exactly the same as g/L or mg/ml. There is no reason to use different units for the same measure for 
different components. The valid use of % is when all components will add up to 100%, which is not the case 
for %w/v. Sigma used to sell a 1 g/ml solution of TCA in water as "100% w/v TCA". I wonder, how 
many people thought 100% meant pure TCA? Wikipedia is not authoritative, but the logic of the following (from 
article on "Volume Fraction") is compelling:

Terminology
'"Percent concentration" does not refer to this quantity. This improper name persists, especially in elementary textbooks. In 
biology, the unit "%" is sometimes (incorrectly) used to denote mass concentration, also called "mass/volume 
percentage." A solution with 1 g of solute dissolved in a final volume of 100 mL of solution would be labeled as "1 %" or 
"1 % m/v" (mass/volume). This is incorrect because the unit "%" can only be used for dimensionless quantities. Instead, 
the concentration should simply be given in units of g/mL.'

We all know that a 1% w/v solution contains 1 g of the component in question, 
per 100 ml of solution. It does not mean that that component makes up 1% of the 
mass of the solution, or 1% of the volume. It does not give us any better 
intuition into the amount of the compound present than would 1 g/100 ml or 10 
g/L. In fact it interferes with intuition, if other components are given in 
mg/ml. In the example above, is the amount of PEG more or less than the amount 
of protein (by weight)? 12.5% peg is 125 mg/ml, so 5-fold excess over protein 
by weight. Suppose you are told that for  a general rule when dissolving 
biological membranes with low-CMC detergents, start off with 1:1 ratio of 
protein:detergent. You want to solubilize your protein at 15 g/L. What should 
the detergent concentration be? Also 15 g/l, but the stock bottle is labeled 
20% w/v. You either convert 15 g/L to 1.5% or convert 20% to 200 g/L before you 
can calculate the volume to add. Not very hard, but for what??

Happy Lunar New Year,
eab



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Re: [ccp4bb] AW: [EXTERNAL] [ccp4bb] Potential weak binding ligand in the active site

[Edward A. Berry]

Happy New Years to all!


delete the ligand you fitted and any water molecules near the ligand and run a 
few rounds of refinement to get an unbiased omit map.


The cavity may be filled with bulk solvent by the refinement program, which can 
reduce the difference signal from whatever is there. If you are using phenix, 
try a polder map (phenix.polder) to avoid this.
eab

On 01/01/2020 07:36 AM, Schreuder, Herman /DE wrote:

Hi Katrin,

First, I think you meant that the green density disappears after contouring at 
6 Sigma and not 6 A?

That you ligand is only partly visible due to disorder and has partial 
occupancy happens often and is no reason for concern. Of course, you could try 
soaking with a 10-fold higher ligand concentration of that would be possible.

The approach you use: fit would you see and don’t fit what you don’t see is 
also correct. However, as you come closer to the noise level of your electron 
density map, you have to be more careful not to introduce model bias and 
artefacts. What I would to copy your current pdb file to another pdb file, 
delete the ligand you fitted and any water molecules near the ligand and run a 
few rounds of refinement to get an unbiased omit map. Then view this map 
together with your refined ligand a see if it makes sense.

Good luck and happy holidays!

Herman

*Von:* CCP4 bulletin board  *Im Auftrag von *Katherine 
Lim
*Gesendet:* Freitag, 20. Dezember 2019 05:57
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [EXTERNAL] [ccp4bb] Potential weak binding ligand in the active site

*EXTERNAL : *Real sender is owner-ccp...@jiscmail.ac.uk 


Hi all,

I apologise in advance for the long post. I am working on solving a structure 
that looks like it could have a ligand bound in the active site. My data was 
obtained from a crystal of just the soluble domain of my protein that had been 
soaked overnight in the ligand solution. The apo crystal structure is already 
known and so I have solved my structure using phaser MR. I have attached the 
Aimless report output of my structure at the end of this email. The current R 
values I have after refinement and adding in all the waters are Rfree: 0.2413 
and Rwork: 0.1897. I can clearly see green density that is much larger (only 
disappears when I contour the Fo-Fc map to about 6 A) than in my control 
crystal that had been soaked in the same concentration of just solvent (I had 
used DMF).

I am struggling to add in my ligand as it doesn't seem like the entire ligand 
can fit in the green density. We think it may be because we have only used the 
soluble domain and so the ligand isn't held very securely since the 
transmembrane domain is missing. I have been trying to fit in smaller sections 
of it and doing an occupancy refinement. So far I have been able to get part of 
the ligand in with an occupancy of about 0.6 but after the refinement run, 
phenix.refine seems to move this part of the ligand slightly out of the area 
where I had tried to fit it into the green density. Interestingly, there isn't 
a big red density in the area that this ligand section has moved to. I would 
appreciate any advice on how I should proceed with trying to figure out if I 
have tried the correct section of the ligand and the kind of refinement 
settings to use with a weak binder.

Space Group



P212121

Unit cell abc



84.76, 89.84, 91.55

unit cell alpha beta gamma



90, 90, 90



OVERALL



LOW RES



HIGH RES

Low res limit



45.78



45.78



1.94

High res limit



1.9



9.11



1.9

Rmerge



0.234



0.052



1.684

Rmeas



0.244



0.054



1.768

Rpim



0.069



0.016



0.527

Total # observations



663073



6282



36851

Total # unique



55174



579



3359

I/sigma



7.5



27.9



1.4

CC ½



0.997



0.999



0.747

Completeness %



99



98.7



94.7

Multiplicity



12



10.8



11

*Katherine Lim *

*PhD Candidate*

*School of Biomedical Sciences; School of Molecular Sciences *

*Marshall Centre for Infectious Disease Research and Training *

*The University of Western Australia *

Re: [ccp4bb] [EXTERNAL] [ccp4bb] Rfree from another mtz file

[Edward A. Berry]

On 09/16/2019 06:29 PM, Mariana Ajalla wrote:

Dear all,

We tried to use the Rfree set from a lower resolution data with a higher 
resolution from the same Crystal. To do so We used aimless at ccp4i with the 
option use free flag from another mtz file and extend the data.

I think it worked, but now we don't know how to be sure we have the same Rfree 
set.
  Does anyone have a way to prove it?
Thank you in advance,
Best,
Mariana



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Mariana,
 If you solve a new dataset with a structure previously refined in an isomorphous crystal, and use 
the same free set as was used for that refinement, you will generally notice that the initial Rfree 
is higher than initial Rwork, as it was with the old dataset. If you start with a new free set, 
Rfree and Rwork will initially be the same, and diverge with refinement. This implies that there is 
some correlation between Fo-Fc calculated with the new data and Fo-Fc from the old dataset. This 
would lead some to say your Rfree is "corrupted" if you choose a new set, and it will 
only be valid after many cycles of refinement to "shake out" the bias. Note that Ian 
Tickle qualified his results by saying it is necessary to refine to convergence for his conclusions 
to hold.

How can the old and new Fo-Fc be correlated, if the errors in the new data are independent from the errors in the old dataset? Well, the errors in measurement  contribute only a small fraction to the Fo-Fc differences, as evidenced by the fact that Rpim is much smaller than either Rfree or Rwork. Thinking in terms of "fitting the noise", if we define the noise to be the differences between Fo and Fc, there are two sources for that noise. One is error in measurement, and as noted these should be completely independent. The other "noise" is the difference between our best-refined structure and the actual structure- modeling solvent, fitting disorder with isotropic or even anisotropic B factors when in fact it is more complicated, single or few alternate conformations when in fact there is a range of positions, etc. If the new data actually represents the same structure, or even a new structure of the same protein in an isomorphous crystal, these errors are likely to be the same in the 
n


ew dataset, and could account for the fact that the fit is better for the 
working reflections which the structure has been refined to minimize against 
the old dataset, when refined against the new dataset.

Again thinking of "fitting the noise", we clearly do not want to fit the noise that comes 
from errors in measurement. But these are uncorrelated between the datasets, so choosing the same 
free set has no effect on this. As for fitting the "noise" that is the difference between 
our model and the real structure, this is a gray area. I tend to think fitting this noise (making 
our structure more like the true structure) would be a good thing, and if it can be biased by using 
all the reflections it would be a good thing. But the fact is it is going to make the R-free lower, 
at least initially, and give you an unfair advantage over the purist who insists on using the same 
free set for isomorphous structures. Until there is a general consensus on this it might be a good 
idea to keep the old free set, if only because your reviewer might be one of those purists!



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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Problem in real space - please sign & invite other scientists to sign this letter

[Edward A. Berry]

Sorry- Richard Muller (How could  I get that wrong?!)



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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Problem in real space - please sign & invite other scientists to sign this letter

[Edward A. Berry]

Agreed. I support green new deal and I think we should be doing all we can to 
combat climate change,
not because I have studied it and concluded it is a problem but because some 
very smart people have, and have come to that conclusion. But I would not sign 
a letter saying that I have concluded, AS A SCIENTIST, that it is a problem we 
can do something about. That turns a scientific investigation into a popularity 
contest. I don't want to be one the 97% that Peter Mueller is talking about in 
his response (currently second in the list, I think) to:
https://www.quora.com/It-is-claimed-that-97-of-climate-scientists-state-that-anthropogenic-climate-change-is-real-What-evidence-do-the-3-who-dont-think-so-have-What-are-some-good-counterarguments-to-their-claims

On 08/20/2019 09:23 PM, Daniel M. Himmel, Ph. D. wrote:

Dear colleagues,

Since when does being a structural biologist make us experts in climatology,

and isn't it a breach of basic ethical practice and professionalism as 
scientists

to sign on as authors to an article for which we have neither contributed

research nor intellectual content of the manuscript?  Are we now going against

the standard to which the editorial policies of leading reputable biological

journals normally hold us as authors?  And doesn't it hurt the credibility

of a serious scientific article, its authors, and the journal in which it 
appears

if biologists with no expertise in earth science/astrophysics appear

without humility as authors to such an article?

Are you not embarrassed to put your name to an article that uses physical

sciences data as a platform for preaching about religion, politics, and economic

theory ("...social and economic justice for all...")?

Does it not upset you when someone unfamiliar with structural biology draws

firm conclusions that heavily depend on the part of a structural model that has 
high

B-factors?  So why are you unconcerned that you may be guilty of an analogous

error when, as structural biologists, you put your name to a controversial 
interpretation

of selected earth science data?  See, for example,

https://blogs.agu.org/geospace/2017/02/24/living-warm-peak-ice-ages/ 

 about the ways

climate data can be misinterpreted by choosing too tight a time interval, and 
lets stick to

structural biology and allied sciences in the CCP4 list, please.

Respectfully,

Daniel M. Himmel


--

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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Better Beamline suggestion!

[Edward A. Berry]

 I would respectfully suggest that higher pixel resolution does not generally help much 
in these situations. If an average spot is 10 or more pixels wide then the profile is 
defined pretty well. But if the spots overlap, they still overlap with higher pixel 
density. It may make profile fitting more accurate, allowing more accurate 
"deconvolution" of the compound spot into its components, but it will not 
improve the overlap. Smaller or better-focused (on the detector, not the crystal?) beam, 
and longer camera length can help.
  It is analogous to chromatography- If two peaks coming off the column 
overlap, collecting smaller fractions will not help to resolve them. It may 
allow a better-informed decision on the cut-off points when you pool the 
fractions, but it won't separate the overlap.
  On the other hand a multi-circle goniometer is very useful. I remember in one 
of our last trips at SSRL (2007-8?) we used a (Huber 4-circle?) and it was very 
easy to have the long axis in the plane of the image throughout the rotation.  
In the absence of such you can resort to carefully bending the loop or bending 
the pin (Jim Holton made a nifty device for bending the pin) while keeping the 
xtal bathed in the cold stream.


On 08/19/2019 11:12 AM, graeme.win...@diamond.ac.uk wrote:

Chandra

What you are looking for here is a beamline with a detector with many pixels 
(so you can resolve the long axis) and a multi-axis goniometer - probably a 
SmarGon / kappa and an Eiger 16M would make a good combination for this. 
Searching on

https://urldefense.proofpoint.com/v2/url?u=http-3A__biosync.sbkb.org_=DwIFAg=ogn2iPkgF7TkVSicOVBfKg=cFgyH4s-peZ6Pfyh0zB379rxK2XG5oHu7VblrALfYPA=VcmIp54F7yM1JdiEMdBdR0y7xinGb-nsn2-3LI_BHto=5VFyqMBHljO-AEcXr3-pqjF8xFyEejXetVFOxOXLp_Y=

Should allow you to make up a short list

Best wishes Graeme

On 19 Aug 2019, at 16:08, Chandramohan Kattamuri 
<1c5b7cb6c764-dmarc-requ...@jiscmail.ac.uk>
 wrote:


Dear All
We recently collected a data set at APS, Chicago with unit cell dimensions of 
68.4; 68.4 and 991.6 A. Our diffraction data extends to 3A with the APS set up, 
however, the long axis has been problematic, resulting in streaking of the 
diffraction data and requires a very specific orientation of the crystal for 
usable diffraction. Can anyone recommend beamlines that can give us higher 
resolution, or a source with a better goniometer allowing for more angle 
manipulation after looping?
Thank a lot
Chandra K






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Re: [ccp4bb] [EXTERNAL] [ccp4bb] [6HR5] collected on an Eiger so Rmerge not relevant

[Edward A. Berry]

I think it is not really the detector, but the strategy. If you decrease 
exposure time by a factor of ten and make up for it by 10 x higher redundancy, 
then obviously R-merge, which is a measure of accuracy of the individual 
frames, is going to suffer. Chi^2 statistics can distinguish this from a real 
problem with the data reduction. And I guess the capabilities of the Eiger 
detectors tend to encourage high-redundancy, low dose/frame strategies? (No 
6HR5 is not mine).

On 07/31/2019 12:49 PM, Weston Lane wrote:

I was looking at the following structure in the PDB:  
https://urldefense.proofpoint.com/v2/url?u=http-3A__www.rcsb.org_structure_6HR5=DwIFaQ=ogn2iPkgF7TkVSicOVBfKg=cFgyH4s-peZ6Pfyh0zB379rxK2XG5oHu7VblrALfYPA=2NZPl2iBIAyGDtZO7h2CtE0DqQqnPQb_sHo94gbPGAo=yti3AVLhix2oCPzcWyto9IuLqgqbU7KWxfTgQ0hleBY=
   I noticed that the R/Rfree stats were pretty high for 2.9A resolution so I followed up by looking for the "Table 1" statistics in the 
journal article.   Link to article: 
https://urldefense.proofpoint.com/v2/url?u=https-3A__www.nature.com_articles_s41589-2D019-2D0311-2D9=DwIFaQ=ogn2iPkgF7TkVSicOVBfKg=cFgyH4s-peZ6Pfyh0zB379rxK2XG5oHu7VblrALfYPA=2NZPl2iBIAyGDtZO7h2CtE0DqQqnPQb_sHo94gbPGAo=qkFehKN3fqEKQJMkmTiecg_OMR82xFIi_5h8yCl-AZs=
   Table is located in the supplemental materials "Table 9".

 From the processing statistics it's clear that the diffraction from that 
crystal wasn't great but I don't want to get hung up on the processing or the 
validity of the structure.  What struck me what this little explanation the 
authors included to explain the outlier statistics in the table:

"Crystal of P36_S1_25 was collected on an Eiger detector, so Rmerge data are not 
relevant."

We all know that Rmerge isn't a great metric for data quality but I've never 
heard that it's detector-dependent.  This doesn't make sense to me.  If it's 
actually true can someone explain, please?

Thanks!

Wes



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Re: [ccp4bb] (EXTERNAL) Re: [ccp4bb] Does ncs bias R-free? And if so, can it be avoided by special selection of the free set?

[Edward A. Berry]

g refinements 
with all 20 working/test sets combinations and averaging the R values).  From 
there on the 'gap' between Rwork and Rfree is a measure of the degree of 
overfitting, so we should really be taking some average of Rwork and Rfree as 
the true measure of agreement (though the biases are not exactly equal and 
opposite so it's not a simple arithmetic mean).  The goal
of choosing the appropriate refinement parameters, restraints and weights is to 
_minimise_ overfitting, not eliminate it.  It is not possible to eliminate it 
completely: if it were then Rwork and Rfree would become equal (apart from that 
small effect from random sampling).

I don't follow your argument about correlation of Fobs from NCS.  Overfitting, and 
therefore CV bias, arises from the _errors_ in the Fobs not from the Fobs themselves, and 
there's no reason to believe that the Fobs should be correlated with their errors.  You 
say "any correlation between the test-set and the working-set F's due to NCS would 
be expected to reduce R-free".  If the working and test sets are correlated by NCS 
that would mean that Rwork is correlated with Rfree so they would be reduced equally!  
There are two components of the Fobs - Fcalc difference: Fcalc - Ftrue (the model error) 
and Fobs - Ftrue (the data error).  The former is completely correlated between the 
working and test sets (obviously since it's the same model) so what you do to one you 
must do to the other.  The latter can only be correlated by NCS if NCS has an effect on 
errors in the Fobs, which it doesn't, or by some other effect such as errors in batch 
scales that are unrelated to NCS.

Overfitting is related to the data/parameter ratio so you don't observe the 
effects of overfitting until you change the model, the parameter set or the 
restraints.  If there were no errors there would be no overfitting and no CV 
bias (actually there would be no need for cross-validation!).

Of course as you say, your tests suggest that there is no CV bias from NCS, in 
which case there's absolutely nothing to explain!

Cheers

-- Ian


On Tue, 4 Jun 2019 at 21:33, Jonathan Cooper 
<0c2488af9525-dmarc-requ...@jiscmail.ac.uk 
<mailto:0c2488af9525-dmarc-requ...@jiscmail.ac.uk>> wrote:

Ian, statistics is not my forte, but I don't think anyone is suggesting 
that the measurement errors of NCS-related reflection amplitudes are 
correlated. In simple terms, since NCS-related F's should be correlated, the 
working-set reflection amplitudes could be correlated with those in the 
test-set, if the latter is chosen randomly, rather than in shells. Am I right 
in saying that R-free not just indicates over-fitting but, also, acts as an 
unbiased measure of the agreement between Fo and Fc? During a well-behaved 
refinement run, in the cycles before any over-fitting becomes apparent, the 
decrease in R-free value will indicate that the changes being made to the model 
are making it more consistent with Fo's. In these stages, any correlation 
between the test-set and the working-set F's due to NCS would be expected to 
affect the R-free (cross-validation bias), making it lower than it would be if 
the test set had been chosen in resolution shells? However, you are always
right and, as you know, I failed to detect any such effect in my limited 
tests. Thanks to you and others for replying.


    On Tuesday, 4 June 2019, 02:07:10 BST, Edward A. Berry mailto:ber...@upstate.edu>> wrote:


On 05/19/2019 08:21 AM, Ian Tickle wrote:
~~~
>> So there you have it: what matters is that the _errors_ in the 
NCS-related amplitudes are uncorrelated, or at least no more correlated than the 
errors in the non-NCS-related amplitudes, NOT the amplitudes themselves.

Thanks, Ian!

I would like to think that it is the errors in Fobs that matter (as may be 
the case), because then:
1. ncs would not bias R-free even if you _do_ use ncs 
constraints/restraints. (changes in Fcalc due to a step of refinement would be 
positively correlated between sym-mates, but if the sign of (Fo-Fc) is opposite 
at the sym-mate, what impoves the working reflection would worsen the free)
2. There would be no need to use the same free set when you refine the 
structure against a new dataset (as for ligand studies) since the random errors 
of measurement in Fobs in the two sets would be unrelated.

However when I suggested that in a previous post, I was reminded that errors in Fobs 
account for only a small part of the difference (Fo-Fc). The remainder must be due to 
inability of our simple atomic models to represent the actual electron density, or its 
diffraction; and for a symmetric structure and a symmetric model, that difference is 
likely to be symmetric.  Whether that difference represents "noise" that we 
want to avoid fitting is another question, but it is likely that (Fo-Fc) will be 
correlated with sym-mates. So I settled for convi

Re: [ccp4bb] Does ncs bias R-free? And if so, can it be avoided by special selection of the free set?

[Edward A. Berry]

en changes which improve the fit to the working reflections would have no 
effect on the values of the free reflections.  It has to go through the structure, 
changes due to refining against the working reflections affect the free reflections, 
which we can call "coupling", and we know that is described by the G-function. 
If free reflections were not coupled to working reflections, Rfree would never change and 
thus would be useless.

For an example, suppose we refine the position of an atom, choosing working reflections 
only in the plane l=0, and free reflections along the l axis (assuming an orthorhombic 
system). The working reflections are only sensitive to position in the x and y 
directions, so the z position would be unchanged by the refinement. But the free 
reflections are only sensitive to position along the z axis, so R-free would be 
unchanged. Presumably the structure would be improved (if that one atom was slightly 
misplaced and all other atoms correctly placed), but the Rfee would not improve. I would 
say this is the direction Chapman and co. were heading with their thin shells of free 
reflections isolated by thick shells of unused guard reflections. If they really succeed 
in eliminating the "bias", then Rfree will be unresponsive to refinement and so 
useless.

Al. et Chapman considered two kinds of coupling- that due to ncs and
direct coupling via Rossmann's G function. They found that choosing free set
in thin shells had little effect, in fact very thick shells with the
test reflections centered in the middle of the shell were required to
significantly reduce the "bias". Now the reciprocal space equivalent of
ncs operators are pure rotational operators, so they relate points in
reciprocal space with precisely the same resolution. Selecting free
reflections in thin shells should thus be sufficient to ensure that
ncs-related reflections have the same free-R flag and avoid bias.  For
my case where ncs is really crystallographic, the shells could be
infinitely thin since the symm-related reflections have precisely the
same resolution. For real ncs the operator takes a reflection to a
non-bragg position which is closely surrounded by reflections, coupled
to them by the G function.
In that case somewhat thicker shells would be required. But using very
thick guard zones around the free reflections implies it is the
G-function they are fighting, as they somewhat implicitly acknowledged by the
discussion of thickness of shells in terms of the radius of the central maximum
of the G function. In that case I wonder if ncs-coupling which still has
to go through G-function coupling to bias a free reflection
contributes significantly compared to the coupling of every reflection to
its direct neighbors.

By using thick guard zones of unused reflections, they end up refining with 
very incomplete data which would be expected to affect the refinement and raise 
the R-free just because the structure is less correct. They control for this by 
refining with another set in which the same number of reflections are deleted 
randomly. But this is not a satisfactory control, because it is generally 
agreed that missing reflections due to an empty zone in reciprocal space is 
more deleterious than missing reflections that are randomly scattered.
Ironically this same "redundancy due to oversampling" that Chapman and co. 
discuss in their introduction allows neighboring reflections to impart most of the 
information of an isolated absent reflection. When the missing reflections are clustered 
together in a thick shell or wedge, a lot of information is not available and the 
structure will suffer. And in particular the structural details that determine structure 
factors in the center of the excluded zone will be poorly determined, since information 
pertaining to them is being excluded. So of course the R-factor calculated from these 
reflections will be higher than with randomly absent data.  Furthermore, if G-function is 
the vehicle by which R-free follows R, R-free will follow less closely and hence 
under-report what improvement is being made.








On Sun, 19 May 2019 at 04:34, Edward A. Berry mailto:ber...@upstate.edu>> wrote:

Revisiting (and testing) an old question:

On 08/12/2003 02:38 PM, wgsc...@chemistry.ucsc.edu 
<mailto:wgsc...@chemistry.ucsc.edu> wrote:
 > ***  For details on how to be removed from this list visit the  ***
 > ***  CCP4 home page http://www.ccp4.ac.uk 
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 > On 08/12/2003 06:43 AM, Dirk Kostrewa wrote:
 >>
 >> (1) you only need to take special care for choosing a test set if you 
_apply_
 >> the NCS in your refinement, eith

[ccp4bb] Does ncs bias R-free? And if so, can it be avoided by special selection of the free set?

[Edward A. Berry]

Revisiting (and testing) an old question:

On 08/12/2003 02:38 PM, wgsc...@chemistry.ucsc.edu wrote:

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On 08/12/2003 06:43 AM, Dirk Kostrewa wrote:


(1) you only need to take special care for choosing a test set if you _apply_
the NCS in your refinement, either as restraints or as constraints. If you
refine your NCS protomers without any NCS restraints/constraints, both your
protomers and your reflections will be independent, and thus no special care
for choosing a test set has to be taken


If your space group is P6 with only one molecule in the asymmetric unit but you instead choose the 
subgroup P3 in which to refine it, and you now have two molecules per asymmetric unit related by 
"local" symmetry to one another, but you don't apply it, does that mean that reflections 
that are the same (by symmetry) in P6 are uncorrelated in P3 unless you apply the "NCS"?


===
The experiment described below  seems to show that Dirk's initial
statement was correct: even in the case where the "ncs" is actually
crystallographic, and the free set is chosen randomly, R-free is not
affected by how you pick the free set.  A structure is refined with
artificially low symmetry, so that a 2-fold crystallographic operator
becomes "NCS". Free reflections are picked either randomly (in which
case the great majority of free reflections are related by the NCS to
working reflections), or taking the lattice symmetry into account so
that symm-related pairs are either both free or both working. The final
R-factors are not significantly different, even with repeating each mode
10 times with independently selected free sets. They are also not
significantly different from the values obtained refining in the correct
space group, where there is no ncs.

Maybe this is not really surprising. Since symmetry-related reflections
have the same resolution, picking free reflections this way is one way
of picking them in (very) thin shells, and this has been reported not to
avoid bias: See Table 2 of Kleywegt and Brunger Structure 1996, Vol 4,
897-904. Also results of Chapman et al.(Acta Cryst. D62, 227–238). And see:
http://www.phenix-online.org/pipermail/phenixbb/2012-January/018259.html

But this is more significant: in cases of lattice symmetry like this,
the ncs takes working reflections directly onto free reflections. In the
case of true ncs the operator takes the reflection to a point between
neighboring reflections, which are closely coupled to that point by the
Rossmann G function. Some of these neighbors are outside the thin shell
(if the original reflection was inside; or vice versa), and thus defeat
the thin-shells strategy.  In our case the symm-related free reflection
is directly coupled to the working reflection by the ncs operator, and
its neighbors are no closer than the neighbors of the original
reflection, so if there is bias due to NCS it should be principally
through the sym-related reflection and not through its neighbors. And so
most of the bias should be eliminated by picking the free set in thin
shells or by lattice symmetry.

Also, since the "ncs" is really crystallographic, we have the control of
refining in the correct space group where there is no ncs. The R-factors
were not significantly different when the structure was refined in the
correct space group. (Although it could be argued that that leads to a
better structure, and the only reason the R-factors were the same is
that bias in the lower symmetry refinement resulted in lowering Rfree
to the same level.)

Just one example, but it is the first I tried- no cherry-picking. I
would be interested to know if anyone has an example where taking
lattice symmetry into account did make a difference.

For me the lack of effect is most simply explained by saying that, while
of course ncs-related reflections are correlated in their Fo's and Fc's,
and perhaps in in their |Fo-Fc|'s, I see no reason to expect that the
_changes_ in |Fo-Fc| produced by a step of refinement will be correlated
(I can expound on this). Therefore whatever refinement is doing to
improve the fit to working reflections is equally likely to improve or
worsen the fit to sym-related free reflections. In that case it is hard
to see how refinement against working reflections could bias their
symm-related free reflections.  (Then how does R-free work? Why does
R-free come down at all when you refine? Because of coupling to
neighboring working reflections by the G-function?)

Summary of results (details below):
0. structure 2CHR, I422, as reported in PDB, with 2-Sigma cutoff)
 R: 0.189  Rfree: 0.264  Nfree:442(5%)   Nrefl: 9087

1. The deposited 2chr (I422) was refined in that space group with the
original free set. No Sigma cutoff, 10 macrocycles.
 R: 0.1767 Rfree: 0.2403  Nfree:442(5%)   Nrefl: 9087

2. The 

Re: [ccp4bb] (EXTERNAL) [ccp4bb] Heme b

[Edward A. Berry]

(in case everyone assumes someone else already answered offline, which they 
probably have)
In coot go to the region where you want to put the heme and do:
File:get monomer  and type HEM in the box that pops up.
This gives plain old heme b (ferroprotoporphrin 9) like in Hb or cyt b
For heme C that would be HEC

On 04/09/2019 07:05 AM, Sanaz Asadollahpour wrote:

Dear all,
How can i build an oxidized HemeB molecule in my COOT structure? do you
have anybody a library from HemeB for me?

best regards...



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Re: [ccp4bb] (EXTERNAL) [ccp4bb] pseudo internal symmetry

[Edward A. Berry]

While I agree with testing in lower symmetry (and I'm sure you have), it is 
also possible that the asymmetry is too slight or doesn't affect the crystals 
contacts, so that the dimer would orient randomly as it packs into the crystal. 
In that case, taking the average over all asymmetric units, the crystal really 
does have the higher symmetry and you would have to use alternate conformations 
as you are doing. I believe a case of this is Mario Amzel's F1 ATPase (pdb 
1MAB) which crystallized in 1H32. The ATPase (ATP synthase we would call it 
today) catalytic subunits are known to form an asymmetric trimer but it 
apparently crystallized with that trimer axis on the 3-fold crystallographic 
axis. The data clearly justified The higher symmetry (according to the 
authors). Later structures with the entire assembly in the asymmetric unit give 
us a good picture of the three different protomers, and it would be interesting 
to go back and try to model it with 3 alternate conformations in each asym
m
etric unit of Amzel's structure.

On 04/03/2019 04:36 AM, Daniele de Sanctis wrote:

Hi all,

we have a structure with a pseudo internal symmetry along a 2-fold axis that 
sits on a 2-fold crystallographic axis. For refinement purposes we have modeled 
the parts that differ with 50% occupancy, but before depositing the structure 
we wanted to make sure that this is the best way to deal with it and it is in 
agreement with PDB standards.

Did anyone deal with similar cases in the past?

Cheers

Daniele


--

ἀρετή
---
Daniele de Sanctis
Structural Biology Group
ESRF - The European Synchrotron
Grenoble, France
Tel 33 (0)4 76 88 2869

http://www.esrf.eu/id29 


--

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Re: [ccp4bb] (EXTERNAL) Re: [ccp4bb] acceptable difference between Average B-factor and Wilson B

[Edward A. Berry]

What if you have one domain with many B-factors aroun 70 and above, and another 
domain with B-factors around 20? The atoms with high B-factor will make 
essentially no contribution to the intensty of spots beyond 3 A, and so have no 
effect on the slope of the Wilson plot byond that. But they will contribute 
mightily to the average atomic B. Or so it seems to me.
eab

On 03/12/2019 04:39 PM, DUMAS Philippe (IGBMC) wrote:


Le Mardi 12 Mars 2019 19:55 CET, Dale Tronrud  a écrit:

Dale
Good to have the opportunity of going back to the crystallography of the 
fifties in these post-modern times...
There is an essential argumentation that should be recalled. The only reason 
for the fact that one ignores low-resolution data in a Wilson plot is that a 
Wilson plot is based precisely upon Wilson statistics, which assumes that the 
atoms are cast randomly in the unit cell.
This assumption obviously does not hold at low resolution and there is no 
reason to obtain a straight line that stems from the latter assumption.
Therefore, I do not think one may say that a Wilson plot tends to ignore atoms 
with high B values.
Consequence: if one has data at rather low resolution, a Wilson plot is 
inherently inaccurate, but if one has data at very high resolution, the Wilson 
plot should give a very decent estimate of the average B and any significant 
discrepancy should ring the bell.
Philippe Dumas



The numeric average of the B factors of the atoms in your model only
roughly corresponds to the calculation of the Wilson B.  While I always
expect the average B to be larger than the Wilson B, how much larger



depends on many factors, making it a fairly useless criteria for judging
the correctness of a model.

While it is pretty easy to understand the average of the B factors in
your model, the Wilson B is more difficult.  Since it is calculated by
drawing a line though the (Log of) the intensity of your structure
factors as a function of the square of sin theta over lambda, it is
rather removed from the atomic B factors.  When drawing the line the low
resolution data are ignored because those data don't fall on a straight
line, and this means that the large B factor atoms in your model are



ignored in the Wilson B calculation.

The Wilson B is (sort of) a weighted average of the B factors of your
model, with the smallest B's given the largest weight.  The actually



weighting factor is a little obscure so I don't know how to simulate it
to adjust the averaging of atomic B's to come out a match.  The easiest
way to compare your model to the Wilson B is to calculate structure
factors from it and calculate the Calculated Wilson B.  No one does this
because it will always come out as a match.  If your calculated Wilson B
doesn't match the observed Wilson B your R values are guaranteed to be
unacceptable and your refinement program will have to be malfunctioning
to create such a model.

If all the B factors in your model are equal to each other, your
refined model will have an average B that matches the Wilson B, because
weighting doesn't matter in that situation.  If you allow the B's to



vary, the difference between the average and the Wilson B will depend on
how high of an individual B factor you are willing to tolerate.  If you
are a person who likes to build chain into weak loops of density, or



build side chains where there is little to no density, then your average
B will be much larger than the Wilson B.  This does not mean there is an
error, it is simply a reflection of the Wilson B's insensitivity to
atoms with large B.

I do not believe comparing the average B to the Wilson B has any
utility at all.

Dale Tronrud

On 3/12/2019 11:34 AM, Eze Chivi wrote:

Dear CCP4bb community,


The average B-factor (calculated from model) of my protein is 65,
whereas the Wilson B is 52. I have read in this BB that "it is expected
that average B does not deviate strongly from the Wilson B". How I can
evaluate if the difference calculated for my data is razonable or not?


Thank you in advance


Ezequiel




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Re: [ccp4bb] (EXTERNAL) Re: [ccp4bb] Question about the electron density maps (.ccp4) in PDBe

[Edward A. Berry]

Also check if the mean value of the map over the ASU is zero. If so, the map 
was probably calculated without the 0,0,0 term of the Fourier series, so zero 
does not represent zero density but rather the mean density.

On 03/04/2019 12:43 PM, Ian Tickle wrote:


Hi Sen

If you multiply the electron density in a voxel by the voxel volume you should 
get an estimate of the number of electrons contained in that voxel, and then 
you can add up the numbers of electrons in all the voxels occupied by an atom 
to get the total number of electrons in that atom, which is basically the same 
as what you are saying.

However note that the electron density is a point sample: it's not the average 
density in the voxel, so the above calculation won't be quite accurate, 
depending on the 'smoothness' of the density.  This is like the error in an 
integral by use of Simpson's rule.  To be sure of accounting for all the 
density you need to sample it finely, say not more that Dmin/4.

There are two more issues here: first the atomic positions are never error-free 
which reduces the contribution to the density by the factor D in the expression 
2mFo-DFc (or mFo for centric phases) for the map coefficients.  So if the 
errors were sufficiently large D would tend to zero and you would get no 
density at all!  The density that you see is really only that part of the true 
density for which there is evidence in the experimental data.

Second, what exactly do you mean by "add all voxel densities around an atom"?  
The electron density could easily extend 2 Ang. from an atomic centre, depending on the 
atom's finite size (represented by the form factor), its thermal motion (B factor) and 
series termination effects (resolution).  So if you don't go out far enough you will fail 
to account for some fraction of the electron count.  The problem is of course you can't 
go so far as to overlap bonded atoms which will be well within 2 Ang. distance.  The 
standard method of dealing with this is to represent 'soft' atoms (where the distance 
between atoms may be less than the sum of their radii) as Voronoi polyhedra (like the 
packing of soap bubbles!).  Is that how you handled it?

Cheers

-- Ian


On Mon, 4 Mar 2019 at 15:47, Yao, Sen mailto:yaosen1...@gmail.com>> wrote:

Hi all,

I have been using the electron density maps available on the PDBe website 
to run some analysis. And I run into this question that I hope that I can get 
some help from the community.

In the ccp4 format, the electron density is represented as a 3-d array map, 
with each number corresponds to the density value of a voxel in real space. If 
I add all voxel densities around an atom together and divided it by the number 
of electrons of that atom, in theory it should give me a ratio with the unit 
Å-3 (angstrom to the power of -3), and this ratio should be inversely related 
to the voxel volume. (Correct me if I am wrong here.) However, after I got this 
ratio for each atom, aggregated it into chains and calculated a median, and 
then compared the chain median to the voxel volume over all PDB structures with 
electron density available, they show a slope of ~1/3 instead of expected 1 
(see attached link). That is, almost all the values in the electron density 
maps are only about 1/3 of represented electrons.

https://drive.google.com/file/d/1K_3uxZfUPuTdH1DtKJgKHLRUA5NHTVPB/view?usp=sharing 



So my question is, is there a conversion or scaling factor that PDBe uses 
to generate the ccp4 files? If so, is that information stored in the ccp4 files 
or anywhere else? And if not, why do I observe this 1/3 ratio pretty 
consistently across the whole PDB?

I would really appreciate any insights on this matter. Thank you!

Sincerely,
Sen

--

Sen Yao, PhD

Center for Environmental and Systems Biochemistry
Markey Cancer Center
University of Kentucky, Lexington KY

Re: [ccp4bb] (EXTERNAL) RE: [ccp4bb] Confused about centric reflections

[Edward A. Berry]

On 03/02/2019 09:59 PM, Ronald E. Stenkamp wrote:

I was taught about centric reflections using different words from those in the 
wiki.

If you look at your crystal structure in projection and the planar view looks 
centrosymmetric, the zone of reflections corresponding to that projection will 
have centric phases, i.e., their phases will be restricted to one of two 
values.  Sometimes those phases are restricted to 0 and 180 degrees, other 
times, depending on the location of the pseudo-inversion center, the phases 
might be 90 or 270 degrees.

So if you look down the two-fold axis in P2, the coordinates of equivalent 
positions become x,0,z and (-x,0,-z).  The zone perpendicular to the two-fold 
contains the h0l reflections, and they end up with restricted phases.  For 
orthorhombic structures, all three zones (h0l, hk0, 0kl) are centric.  And if 
you look at trigonal structures, as in P3(1), there are no centric reflections. 
 (In P3(1)21, there are centric zones, but they aren't the hk0 reflections.

Ron Stenkamp


Thanks, I think I see that. Any time you have a two-fold axis, proper or screw, the 
projection of density onto a plane perpendicular to that axis and passing through the 
origin will be 2-fold symmetric. Any reflection whose scattering vector lies within that 
plane will be taken to its Friedel mate by the reciprocal space version of the operator, 
and that reflection's scattering vector will lie along the same line but in the opposite 
direction. The further projection of the density onto one of those scattering vectors 
will obey the 2-fold and be centrosymetric. Centrosymmetry in one-dimension is also 
called "even function" (vs odd function). The fourier components of even 
functions are non-zero only for the cos terms (sin is an odd function), taking zero at 
the point of centrosymmetry. If the center of symmetry is offset from the origin then 
there is a phase shift equal to 2pi times the fractional distance from origin to center 
along the scattering vector. If we don't like negative am
p
litudes, we make them positive and add pi to the phase. So we get 2 possible 
phases, separated by 180*. (Or something like that.)



-Original Message-
From: CCP4 bulletin board  On Behalf Of Edward A. Berry
Sent: Saturday, March 2, 2019 2:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Confused about centric reflections

The wiki:

https://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Centric_and_acentric_reflections

says:
"A reflection is centric if there is a reciprocal space symmetry operator which 
maps it onto itself (or rather its Friedel mate).
. . .
Centric reflections in space group P2 and P21 are thus those with 0,k,0."

The operator -h,k,-l does NOT take 0,k,0 to its Friedel mate.
it takes h,0,k to their Friedel mates. In other words the plane perpendicular 
to the 2-fold axis, at 0 along the axis

Or am I missing something?
eab



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Re: [ccp4bb] Confused about centric reflections

[Edward A. Berry]

Thaks! typo- h,0,l not h,0,k
I have not registered for editing on that wiki,
so I was hoping someone else would take care of it.
But it seems all that's needed is to confirm your account, so I'll register and 
give it a try. Hope I don't make things worse!
(what is this urldefense.proofpoint? did my institution add that, or JISCMAIL?)


On 03/02/2019 05:28 PM, Dale Tronrud wrote:

You are correct, other than your typo.  The centric zone in a
monoclinic space group (B setting) is h0l.

This web site is a wiki so you should be able to correct it yourself.

Dale Tronrud

On 3/2/2019 2:00 PM, Edward A. Berry wrote:

The wiki:

https://urldefense.proofpoint.com/v2/url?u=https-3A__strucbio.biologie.uni-2Dkonstanz.de_ccp4wiki_index.php_Centric-5Fand-5Facentric-5Freflections=DwICaQ=ogn2iPkgF7TkVSicOVBfKg=cFgyH4s-peZ6Pfyh0zB379rxK2XG5oHu7VblrALfYPA=HENAQrgItEIhnnUZhWe8GMKW5sRX2v7V-rsby0AB7LQ=8D9Gvr-8AQeITEdUbf2xeIBDNzdTzUTnE78AI70O1J0=


says:
"A reflection is centric if there is a reciprocal space symmetry
operator which maps it onto itself (or rather its Friedel mate).
. . .
Centric reflections in space group P2 and P21 are thus those with 0,k,0."

The operator -h,k,-l does NOT take 0,k,0 to its Friedel mate.
it takes h,0,k to their Friedel mates. In other words the plane
perpendicular to the 2-fold axis, at 0 along the axis

Or am I missing something?
eab



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[ccp4bb] Confused about centric reflections

[Edward A. Berry]

The wiki:

https://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Centric_and_acentric_reflections

says:
"A reflection is centric if there is a reciprocal space symmetry
operator which maps it onto itself (or rather its Friedel mate).
. . .
Centric reflections in space group P2 and P21 are thus those with 0,k,0."

The operator -h,k,-l does NOT take 0,k,0 to its Friedel mate.
it takes h,0,k to their Friedel mates. In other words the plane
perpendicular to the 2-fold axis, at 0 along the axis

Or am I missing something?
eab



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Re: [ccp4bb] visualising anis B factors

[Edward A. Berry]

(Another idle post to see if we can hit the record)

One assumes the image is an attachment, and won't be included in the reply.
But no, its inline with the text. Easy enough to delete if you notice that,
but less likely to be noticed as the thread gets longer and longer . . .

On 01/23/2019 08:26 PM, Paul Emsley wrote:

I nominate this as the most annoying thread in all of CCP4BB history.

Stop including the image already! Just stop!



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[ccp4bb] What to check in annotated pdb before approving release? was Very strange LINK cards appearing in recent structures

[Edward A. Berry]

So, are there any other common errors of annotation that one should look for?
I've just submitted seven structures (same protein) and there are some
newly introduced LINK records, but all seem correct.
eab

On 11/11/2018 02:31 PM, Robbie Joosten wrote:

Hi Tristan,

Erroneous LINK records happen quite a lot and used to be the combination of 
aggressive annotation software and depositors not paying attention to the 
comments from the annotators. They make up a large fraction of the bug reports 
I have sent to the PDB over the years. They are usually fixed very quickly by 
the annotators, as long as someone takes time to report them.

This case looks like an error in a refinement program which nevertheless should 
have been caught by the depositors. What I would like to know is whether the 
deposited, pre-annotation model had the LINKs or not.

LINKs are a bloody nightmare when it comes to annotation. At the moment there 
is no record keeping of targets and chemical modifications in a dictionary on 
the side of the PDB so there is also no standardisation. IMO mmCIF makes it 
easier to store the restraints with the coordinates, but there is still no neat 
mapping by LINK identfiers the way the LINKR format works in Refmac. I think 
that is a missed opportunity.

Sorry for the rant, I blame the F1.

Cheers,
Robbie


Op 11 nov. 2018 19:47 schreef Tristan Croll :

I've seen instances like the following in roughly half a dozen deposited
structures over the past year or so. Each time I've contacted the
authors, who've been just as mystified as me by them - and certainly
didn't add them on purpose. It seems to me that some fairly
commonly-used package is erroneously turning clashes into LINK cards in
some circumstances. I just found the following clearly wrong LINKs in
6caj (deposited January this year):

LINK CD2 PHE I 266 CG2 THR I 272 1555   1555
   1.47
LINK CE2 PHE I 266 CG2 THR I 272 1555   1555
   1.47

... which looks like the attached image. The same bonds are also
specified in the mmCIF file, for the record.

Anyone have any clue?

Best regards,

Tristan



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Re: [ccp4bb] VERY old mtz file..

[Edward A. Berry]

In case one wants to play around with openVMS or just recover some 
now-unreadable old data,
and doesn't have a vax farm or even a single vax computer,there are 
step-by-step instructions for setting up a vax system using the simh emulator:
http://www.wherry.com/gadgets/retrocomputing/vax-simh.html
Simh is under active development and I have no idea if those old instructions 
would work with the latest nightly build (but they should). The mailing list 
is: http://mailman.trailing-edge.com/mailman/listinfo/simh

I did this back around 2004 and it worked very well. As described, DEC (then compaq and I 
think now HP) provides a free "hobbyist" license and a cdrom with the operating 
system and standard software for networking, fortran and C compilers. Using the 
instructions I was able to get networking working, could FTP things between the vax and 
other computers, and set up an nfs-served disk that both vax and host could read and 
write. I was able to make tape images from vaxbackup tapes using dd (including tapes with 
multiple savesets) and mount them on the emulator. Doing this I was able to recover old 
vax email and, using the vax as mail server and mozilla as client, transfer all the old 
email to a mozilla-readable format which I can access today using seamonkey or 
thunderbird. More importantly I was able to recover old data saved from ssrl vaxen on 
exabyte tapes.

It would be nice if ccp4 would make an old release available on a "hobbyist" 
license for use on vax (although coming to my senses I can't imagine what use anyone 
would have for such a thing - could be a real waste of time!).

This was about the time that support for lcbvax at LBL chemical biodynamics 
division was ending, and I was able to resurrect it from standalone backup 
tapes and keep it running for a few die-hard vax users for a few years.

If you just want to recover old data (and have a compatible tape drive), you 
can just use the vmsbackup utility (described by Kay Diederichs in an earlier 
post). It loses most of the meta-data, but restores diffraction images that 
work fine with the HKL package. But the emulator is much more fun!
eab

On 11/14/2018 05:15 AM, Harry Powell wrote:

Hi

Weren't the CCP4 base-level routines re-written from FORTRAN to C sometime in 
the late 1990's? Very occasionally I used to find bugs that had been introduced 
in this process (or possibly not corrected...) so it's possible that Eleanor's 
file might be readable with a really old code base.

BTW, I have recently had cause to search out a VMS system and have found 
someone with a whole farm of running VAXes (or VAXen if you're geeky).

Harry

On 14 Nov 2018, at 09:46, Ian Tickle wrote:


The CCP4 routines for MTZ and map files are written in C and thus do not use a 
Fortran unformatted OPEN statement, they use C-style block read & write.

Cheers

-- Ian


On Wed, 14 Nov 2018 at 08:59, Kay Diederichs mailto:kay.diederi...@uni-konstanz.de>> wrote:

It is not necessary to do error-prone conversions manually: the ifort 
Compiler understands the convert='VAXD' Option in its OPEN statement - see

https://software.intel.com/en-us/fortran-compiler-developer-guide-and-reference-open-convert-specifier

Thus one could just write a tiny read-write loop.

HTH
Kay

On Wed, 14 Nov 2018 00:51:02 +, Zhijie Li mailto:zhijie...@utoronto.ca>> wrote:

>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 mailto:CCP4BB@JISCMAIL.AC.UK>> on 
behalf of Zhijie Li mailto:zhijie...@utoronto.ca>>
>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 

Re: [ccp4bb] R-merge is too high !!

[Edward A. Berry]

The fact that chi^2 is approximately 1.0 in all shells says that the deviations 
are about what is expected from the error model. The fact that Rpim is much 
lower than Rmeas means that you have rather high redundancy. This would seem to 
be a case of collecting low dose per image and making up for it with high 
redundancy, a strategy that has been recommended to ensure a full dataset even 
in the case of high radiation sensitivity.  In my opinion the high Rmerge is 
nothing to worry about. Look instead at CC1/2 and I/sigI which seem fine.

On 09/28/2018 04:09 AM, Zhang Foggy wrote:

Dear All,

Sorry for the off-topic.

I recently collected a set of data. The diffraction spots are extremely sharp. 
However, When I used HKL3000 to scale it, I get a final resolution at 3.1A with 
overall R-merge ~0.54 (R-merge in the highest 3.2A-3.1A shell: 1.59). Then I 
solve the structure with final R value 0.19 and R free value 0.24 although I 
know this Rmerge value is totally unacceptable, and the density looks perfect.

I also tried to collect other four set of data with different crystals. 
unfortunately, all of them have same problem.

I ask one of my friend who is an expert in HKL3000, but he had no idea about 
it. Does anyone has suggestions?

Here is the scale information for your review:
Space group: P43 (I also tried P1, the Rmerge value is still similar)

Shell Lower Upper Average  Average Norm. Linear Square
  limitAngstrom   I   error   stat. Chi**2  R-fac  R-fac  Rmeas   Rpim  
CC1/2CC*
   50.00   6.6711.6 0.9 0.3  1.165  0.191  0.284  0.198  0.052  
0.975  0.994
6.67   5.30 4.5 0.5 0.3  0.952  0.317  0.313  0.329  0.086  
0.971  0.993
5.30   4.63 7.3 0.7 0.5  0.961  0.293  0.297  0.304  0.081  
0.975  0.994
4.63   4.21 7.0 0.8 0.6  0.986  0.369  0.358  0.382  0.101  
0.969  0.992
4.21   3.91 5.6 0.8 0.6  1.040  0.522  0.491  0.541  0.142  
0.955  0.988
3.91   3.68 4.6 0.9 0.7  1.064  0.718  0.669  0.746  0.203  
0.929  0.981
3.68   3.49 3.5 0.9 0.8  1.092  1.059  0.986  1.101  0.299  
0.882  0.968
3.49   3.34 2.6 0.9 0.8  1.092  1.382  1.298  1.438  0.395  
0.829  0.952
3.34   3.21 2.1 0.9 0.8  1.084  1.543  1.489  1.614  0.468  
0.772  0.933
3.21   3.10 1.6 0.9 0.8  1.070  1.591  1.669  1.680  0.529  
0.645  0.885
   All reflections  5.0 0.8 0.6  1.048  0.538  0.487  0.559  0.153

Thank you.

Liang


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Re: [ccp4bb] [ccpem] Creating map volume of custom size

[Edward A. Berry]

Hi Melanie,
Mapmask was suggested. The XYZLIM command of mapmask takes values in grid 
points or fractional coordinates.
You could make a 300A cube by giving fractional coordinates of 300/(current 
cell edge),
But since that doesn't change the grid it won't change the thickness of your 
slices.
I don't think mapmask (old name "extend") can change the grid.

Maprot was also mentioned, and I think this should work. You need to generate a 
300x300x300 target map (WORKIN) and perhaps a mask (could be your entire 
current cell). Use the identity rot-trans operator, or with some non-zero 
translation if necessary to get your model entirely in one cell.

But it might be much easier to just recalculate your map from structure factors 
with equal grid spacings using fft.
So if you want half-angstrom slices, set the grid values (nx,ny,nz) to twice 
the cell dimensions in Angstroms.
The FFT has some restrictions on the grid so you may not be able to choose 
exactly what you want, but could get pretty close to even spacing. (In fact 
programs that write maps tend to make approximately equal spacing, like 1/2 or 
1/3 of the resolution, by default- so it would be surprising if your current 
map has drastically different spacing along the different axes).  If you expand 
your data to P1 before fft, then the only restrictions are that nz be an even 
number (and none of the nx,ny,nz have prime factors greater than 19). You 
should be able to get very nearly equal spacing with that.

I wonder if there is any program that uses conventional FT and allows arbitrary 
grid choices?
eab


On 07/16/2018 11:28 AM, melanie.voll...@diamond.ac.uk wrote:

Thanks everyone who replied to my post. I got a wealth of ideas.


Melanie

Hello everyone,


I have a question about manipulating map files.


I want to use my map derived from X-ray diffraction and create a standard 
volume for it, say 300A3.

This is to be able to create slices of equal dimensions along all three 
directions of the electron density stored in the map file. Currently my slices 
differ in dimensions as the default volume is defined by the unit cell.


I don't think there is anything in the map tools from the X-ray field I know. I 
had a play with Chimera and I think there is a tool in there to use some form 
of reference cell created from coordinates. But I don't want to make use of a 
coordinates file.


I just want to inflate my unit cell to a standard volume.


Any ideas would be very welcome.


Thanks


Melanie

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Re: [ccp4bb] To post to ccp4bb...

[Edward A. Berry]

Is it a membrane protein?
Looks like a phospholipid without the phosphate. diglyceride maybe?
But I see not much blue density. Presumably this fills out if you contour
at a lower but still reasonable level?

On 07/02/2018 06:26 AM, Christopher Horne wrote:

Does anyone have any insight into this weird density?

Present at dimer interface, PEG in condition. I have seen a previous post which mentions "PEGs 
can create ugly "snakes" of variable density that may be challenging to model".



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Re: [ccp4bb] Regarding symmetry notion in coot

[Edward A. Berry]

On 06/21/2018 03:34 PM, John Berrisford wrote:

 From a PDB deposition point of view, I echo Paul’s comment. If possible, 
please move your molecule closer to the origin. It helps with the curation 
process and is easier for users of your entry as some software struggles with 
such extreme transformations.

Regards

John


Now that we've all read the manual :),
Since the origin shift in question here is always integral: { 1 0 1 }
It seems this is not about _where_ in the unit cell, but in _which_ unit cell.

As I understand it the symop: -X, Y+1/2, Z + (0 -1 1)
is what would work if you were in the first cell { 0 0 0 }
So you need to
Take the current coordinate X =x,y,z
subtract the origin translation O = { 1 0 1 },
apply the symop S = -X, Y+1/2, Z + (0 -1 1) = -X,Y,Z +(0,-1/2,1)
add back the origin translation O

something like X' = S(X-O) + O

which could take three runs of pdbset.

But you could really work out an operator that would do it in one run:
the symop S consists of a rotation and translation

S(x) = M(x) + T
S(X-O) = M(X-O) +T
S(X-O) = M(X) - M(O) +T
X' = S(X-O) + O = M(X) - M(O) + T + O

In the example given, M = -X,Y,Z; T = (0,-1/2,1); O = (1 0 1)
M(O) = -1,0,1
-M(O) = 1,0,-1
add T gives 1,-1/2,0
add O gives 2,-1/2,1
so the operator should be
-X+2,Y-1/2,Z+1
or something like that, which you could use in pdbset.
Matrix inverts along x, coordinates are way positive in x, result way negative,
  need to add 2 unit cell translations to position it adjacent to orig 
coordinates

Would something like that work?


*From:*CCP4 bulletin board  *On Behalf Of *Paul Emsley
*Sent:* 21 June 2018 17:30
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] Regarding symmetry notion in coot

On 20/06/18 20:49, Anurag Misra wrote:

What is the meaning of the last field — the one in curly brackets -- that

describes the symmetry transformation of a given molecule in coot?


See Section 4.11 of Coot User Manual: "Symmetry"


For example, for

a given X Y Z, coot displays the symmetry transformation of an equivalent

position as " -X, Y+1/2, Z + (0 -1 1) & { 1 0 1 }”.

Can the symmetry molecule be written like bellow by summing respective 
values?


In the general case, no. Move your molecule closer to the origin - so that the 
pre-translation is (0,0,0) - then coot will report "conventional" symmetry.

Regards,

Paul.

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Re: [ccp4bb] Problems with HEC in CCP4

[Edward A. Berry]

This is interesting. If the parameters for heme C reflect the molecule before 
linking
to cysteines in the protein, and the modifications will be made as specified by
link information in the dictionary, then actually heme C (HEC) should be the 
same
as HEM (heme b, protoheme). HEC is superfluous, unless its definition could
include the fact that it must be covalently linked.

Heme C is a regular heme like in myoglobin (heme B, protoheme) that has been 
attached to a protein via two cysteines. Each Cys Sg adds across the double 
bond in a vinyl group, the latter consisting of CA connected to ring by single 
bond, CB connected to CA by double bond. Cys Sg attaches to the CA atom, and 
the double bond becomes single.
However comparing the .cif library files, the double bond in HEC has moved from 
CA=CB to ringC3=CA:
(third letter B or C in the atom names indicates which vinyl group it is in)

ccp4-7.0/lib/data/monomers/h/HEM.cif:
HEM  CABC3B   single  1.4740.020
HEM  CBBCAB   double  1.3300.020

HEM  CACC3C   single  1.4740.020
HEM  CBCCAC   double  1.3290.020

ccp4-7.0/lib/data/monomers/h/HEC.cif:
HEC  CABC3B   double  1.4830.020
HEC  CBBCAB   single  1.5100.020

HEC  CACC3C   double  1.4830.020
HEC  CBCCAC   single  1.5100.020

And when I look at a protein containing HEC in coot, it displays a double bond 
from ring to CA.
(By the way in coot "get monomer" HEC comes up with one of the pyrrole rings 
flipped 90* from the porphyrin plane!)

I suggest that either:
 HEC should be exactly like HEM, and the link definition would include: link SG 
to CA, add H to CB, and change the double bond to single;

Or, HEC should be pre-hydrogenated: vinyl is ethyl, and the link definition 
just involves removing H from CA and making the link SG-CA.

eab



If the parameters for heme c reflect themolecule before linking
to cysteines in the protein, and the modifications will be made as specified by
link information in the dictionary, then actually heme C (HEC) should be the 
same
as heme b (protoheme).

On 05/15/2018 04:35 AM, Eugene Osipov wrote:

Dear CCP4 developers,
please fix errors with heme c dictionary file HEC.cif.
Edward Berry described this problem in detail 4 years ago:
https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg36393.html
This error already affects deposited entries, e.g.: 4QO5 clearly contains 
errors in vynil groups of heme c.


--
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com <mailto:e.m.osi...@gmail.com>

Re: [ccp4bb] Problems with HEC in CCP4

[Edward A. Berry]

Does this mean- if the pdb file has standard PDB link records involving HEC, 
like
LINK SG  CYS A  32 CAB HEM A  93 1555   1555  1.82
LINK SG  CYS A  35 CAC HEM A  93 1555   1555  1.81
then refmac will look up the necessary modifications in the ccp4 dictionary?
Or do we need to tell refmac to apply those mods?
eab

On 05/15/2018 05:00 AM, Robbie Joosten wrote:

Addendum:

4qo5 actually lacks the right LINK records altogether. It will only work if you 
add these by hand. Perhaps you should ask the depositors or the PDB to add the 
appropriate LINK records.

Cheers,
Robbie


-Original Message-
From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of Robbie
Joosten
Sent: Tuesday, May 15, 2018 10:51
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Problems with HEC in CCP4

Hi Eugene,

The problem is not in the HEC.cif file but with the LINKs which modify the
chemistry. These were fixed a while ago and the LINKs were added to the
CCP4 dictionary. I thought these were released already. If not, they will be
soon.

Cheers,
Robbie


-Original Message-
From: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> On Behalf Of

Eugene

Osipov
Sent: Tuesday, May 15, 2018 10:36
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Problems with HEC in CCP4

Dear CCP4 developers,
please fix errors with heme c dictionary file HEC.cif.
Edward Berry described this problem in detail 4 years ago:
https://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg36393.html
This error already affects deposited entries, e.g.: 4QO5 clearly
contains errors in vynil groups of heme c.


--

Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com <mailto:e.m.osi...@gmail.com>

Re: [ccp4bb] Compound with flexible conformation but nM Kd

[Edward A. Berry]

For proteins in membranes, or proteins purified in the presence of detergents 
and/or lipids, the active site is sometimes surrounded by a hydrophobic mileau. 
The actual concentration that the binding site sees is then dependent on 
partitioning of the ligand between the water (where its concentration for Kd is 
determined) and the hydrophobic phase. Hydrophobic substituents could then 
result in higher concentrations at the binding site, and thus lower the 
apparent Kd, while having nothing to do with binding.

On 04/26/2018 10:50 AM, WENHE ZHONG wrote:

Dear Community,

A little bit out of topic here. We are applying the structure-based approach to 
design compounds that can bind our protein target. We have synthesized a series 
of analogues based on the same scaffold with different substituents at one 
particular site. The most potent analogue (nM Kd) has a long alkyl chain 
substituent. We thought this hydrophobic substituent should have strong 
interactions with the target protein leading to nM range affinity. However, 
crystal structures show very weak densities for this substituent and no obvious 
interaction between the substituent and the target protein, suggesting that 
this long alkyl chain substituent is flexible without binding to the protein. 
This binding site is relatively negative charged according to the electrostatic 
potential analysis.

So it is a puzzle to me that how this dynamic and hydrophobic alkyl chain 
substituent can lead the compound to achieve nM affinity (>10-fold better than 
any other substituent) — in particular the binding site is not hydrophobic and no 
interaction is found between the substituent and the protein.

Anything I have miss here that can increase the binding affinity without 
interacting with the target?

Thanks.

Kind regards,
Wenhe

Re: [ccp4bb] How to get Imean/SIGImean from separated anomalous data?

[Edward A. Berry]

Thanks, that seems to work also, and with lots of nice graphical analysis.
With Task "Import merged data", or all in one go with "convert intensities to amplitudes" 
selecting the .cif file as input and option "output Imean for anomalous data", to run ctruncate on 
the result.

But I have a long way to go to get used to I2. It was disconcerting not being 
able to set the output filenames, or even find the output files except by 
listing files in the project directory.  I guess it's a new paradigm, where you 
don't need to worry about details like file names and you identify datasets by 
internal labels within the gui. And going back to the finished job, looking at 
input and options, there was very little information about exactly what had 
been done (compared to the detailed forms in ccp4i). But now I am motivated to 
learn the new system!
eab

On 03/14/2018 06:42 AM, Eleanor Dodson wrote:

No problem with GUI2..

Eleanor

On 13 March 2018 at 22:38, Edward A. Berry <ber...@upstate.edu 
<mailto:ber...@upstate.edu>> wrote:

Thanks- that works!
pdb-redo/tools/cif2cif in.cif out.cif
eab


On 03/13/2018 05:47 PM, Robbie Joosten wrote:

Hi Ed,

The pdb-redo program cif2cif does that. I implemented this feature 
because there was no obvious CCP4 program that did this. You have to have your 
data in mmCIF format though.

Cheers,

Robbie

Sent from my Windows 10 phone




-
-

*From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Edward A. Berry <ber...@upstate.edu <mailto:ber...@upstate.edu>>
*Sent:* Tuesday, March 13, 2018 10:25:25 PM
*To:* CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
*Subject:* [ccp4bb] How to get Imean/SIGImean from separated anomalous 
data?

I want to work with an mmcif file that has I+,sigI+,I-,SigI-;
in different columns for each reflection (no Imean).
cif2mtz gives those same columns in the output mtz, with or without 
ANOMALOUS keyword.
cif2mtz and truncate seem to need the anomalous data as separate 
reflections hkl and -h-k-l in order to calculate Imean or Fmean. What is the 
best way to do this?
Thanks,
eab

Re: [ccp4bb] How to get Imean/SIGImean from separated anomalous data?

[Edward A. Berry]

Thanks- that works!
pdb-redo/tools/cif2cif in.cif out.cif
eab


On 03/13/2018 05:47 PM, Robbie Joosten wrote:

Hi Ed,

The pdb-redo program cif2cif does that. I implemented this feature because 
there was no obvious CCP4 program that did this. You have to have your data in 
mmCIF format though.

Cheers,

Robbie

Sent from my Windows 10 phone

--
*From:* CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> on behalf of Edward A. Berry 
<ber...@upstate.edu>
*Sent:* Tuesday, March 13, 2018 10:25:25 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] How to get Imean/SIGImean from separated anomalous data?
I want to work with an mmcif file that has I+,sigI+,I-,SigI-;
   in different columns for each reflection (no Imean).
cif2mtz gives those same columns in the output mtz, with or without ANOMALOUS 
keyword.
cif2mtz and truncate seem to need the anomalous data as separate reflections 
hkl and -h-k-l in order to calculate Imean or Fmean. What is the best way to do 
this?
Thanks,
eab

[ccp4bb] How to get Imean/SIGImean from separated anomalous data?

[Edward A. Berry]

I want to work with an mmcif file that has I+,sigI+,I-,SigI-;
 in different columns for each reflection (no Imean).
cif2mtz gives those same columns in the output mtz, with or without ANOMALOUS 
keyword.
cif2mtz and truncate seem to need the anomalous data as separate reflections 
hkl and -h-k-l in order to calculate Imean or Fmean. What is the best way to do 
this?
Thanks,
eab

Re: [ccp4bb] questionable structures

[Edward A. Berry]

On 01/21/2016 05:48 PM, Edward A. Berry wrote:



Also, pubmed commons allows comments on the abstract page for every article.
Whenever you look up an article on pubmed, there is an invitation to
leave comments below, and if a comment is left it is prominently
displayed above the article like "See comment in PubMed Commons below"
For example
http://www.ncbi.nlm.nih.gov/pubmed/7737189

This could also be used a warning to the unwary
that something may not be quite right -
if you are not afraid of being sued.


Now NCBI is discontinuing this capability due to lack of participation:
https://ncbiinsights.ncbi.nlm.nih.gov/2018/02/01/pubmed-commons-to-be-discontinued/
 .
If you think it is a good service even though only a small percentage
of papers get any comments, you can say so by leaving a comment on that blog 
post
(there are quite a few already).


eab

On 01/21/2016 03:19 PM, Bellini, Dom wrote:

Perhaps a temporary and quick solution could be to have an online spreadsheet 
where every one could annotate erroneous structures when they encounter one 
(writing down PDB code with a comment).


It wont solve publication-related issues but at least it would help people 
working with PDB big data to filter out the ones on the list from their 
databases.


I am taking bets on how long the list will be!


D




--
*From:* CCP4 bulletin board <CCP4BB@jiscmail.ac.uk> on behalf of Bernhard Rupp 
<hofkristall...@gmail.com>
*Sent:* 21 January 2016 19:56
*To:* CCP4BB@jiscmail.ac.uk
*Subject:* Re: [ccp4bb] questionable structures
Simple statement:  "The structure model is is almost certainly wrong (to the point 
of 'beyond reasonable doubt') and it should not be in any data base."
How to handle the rest of the paper depends on the degree of inference based on 
the flawed model. But I am not doing all the work for the editors ;-)

BR

On Thu, Jan 21, 2016 at 8:40 PM, Keller, Jacob <kell...@janelia.hhmi.org 
<mailto:kell...@janelia.hhmi.org>> wrote:

BR:

__ __

Not clear what more you would have wanted to the editor to write—what’s 
missing?

__ __

Or were you commenting on the lack of concrete actions?

__ __

JPK

__ __




--
-
Bernhard Rupp (Hofkristallrat a. D)
001 (925) 209-7429
+43 (676) 571-0536
b...@ruppweb.org <mailto:b...@ruppweb.org>
hofkristall...@gmail.com <mailto:hofkristall...@gmail.com>
http://www.ruppweb.org/
-
The hard part about playing chicken
is to know when to flinch
-

Re: [ccp4bb] How to define the DNA bond between P and O3’ from two different symmetric units?

[Edward A. Berry]

Dear Peng,
The choice of asymmetric unit is somewhat arbitrary. Please forgive me and 
ignore this if I am saying something obvious and you already know this does not 
apply in your case, but I see most of your previous posts concern EM 
structures, so perhaps you are relatively new to solving x-ray structures?

I assume that you are looking at your structure in coot (or similar), and you 
have turned on the option to show symmetry mates, and you found two places 
where your principal model ends but is continued by a symmetry mate.  If a 
portion of the molecule is visible in symmetry mates, that means it is present 
in your principal model but in a different place- in other words you have built 
parts of two separate molecules.  This is a common result. So you want to find 
out which residues they are, delete them from your main model, and copy then 
from the adjoining symmetry molecule into your main model. You may have to do 
this twice, if your longest stretch is 6BP and there should be 20. But bear in 
mind the second place you observed may be symmetry related, and disappear after 
you fix the first. Possibly some of the molecule is disordered.

There is probably a way to do this all in coot, using things like copy-fragment 
and merge-molecules, but I am a beginner in coot. What you can do is:

Find out which residues you want to transfer from the symmetry mate to main 
model

file:save symmetry coordinates, click on an atom in the symmetry mate where it 
abuts, and give a filename to save it as.

Use a text editor to replace those atoms in your main model with the atoms from 
the symmetry mate (save with a new name just in case)

Load into coot and see if it looks OK

Refine the new model and see if you get essentially statistics equal to before 
and no clashes

The achesymm web server (http://achesym.ibch.poznan.pl/) might be able to do 
this automatically.

(the blind leading the blind - always glad to hear better solutions)

On 01/30/2018 05:41 PM, Peng wrote:


Dear Nicolas,
Thank you for your help.
Our DNA is not a palindromic sequence, and I think it should not be degraded 
easily.
The data can be processed in the space groups C2221, P41212 or lower, with 
12bp, 6bp ,24bp or longer in one NCS. I can see the connection between two 
symmetric DNA molecules in all these space groups.
Actually, I am really confused about that because 20pb-DNA was used in 
crystallization.
Peng




在2018年01月30 16时58分, "Nicolas FOOS"写道:


Dear Peng,

to me your problem sound a bit strange, except if it's a  palindromic 
sequence. I don't understand how you can have one part  of the DNA in one 
asymmetric unit and one in another one. My  question are : maybe you 
considering a NCS as a true symmetry and  underestimate the unit-cell 
dimensions? Or you DNA has been  degraded by DNase and the current size is 
not 20pb, in this case  you are not supposed to create an artificial  
connection. Is the  resolution good enough to be certain of the sequence ?

Sorry, I don't provide any answer, but I am curious and try to  
understand what is going on.

Hope this finally help.

Nicolas

Nicolas Foos
PhD
Structural Biology Group
European Synchrotron Radiation Facility (E.S.R.F)
71, avenue des Martyrs
CS 40220
38043 GRENOBLE Cedex 9
+33 (0)6 76 88 14 87
+33 (0)4 76 88 45 19

On 29/01/2018 20:28, Peng wrote:


Hello, everyone，

 Recently, we solve a protein-DNA complex.20bp-DNA was used for 
crystallization, but only 6bp wasfound in one symmetric unit.

My question is：

How to define the DNA bond between P and O3’from two different 
symmetric units during my refinement?

Peng

Re: [ccp4bb] AW: Re: [ccp4bb] AW: Re: [ccp4bb] coordinate transformation

[Edward A. Berry]

qu...@jiscmail.ac.uk 
<mailto:176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk>> wrote:

I showed you pdbset ..

Find the centre of mass for your assembly.

Move it where you will

pdbset xyzin mow.pdb

end

Find  CoM 0.7 1.3 -0.2

Hmm - a little thought - centre at 1 -1 0   say

pdbset yzin now.pdb xyzout changed.pdb

symgen x , y-2, z

    end

New CoM  0.7 -0.7  -0.2

Eleanor

On 18 December 2017 at 00:19, Edward A. Berry <ber...@upstate.edu 
<mailto:ber...@upstate.edu>> wrote:

Neat idea!
And do you have a 1-line command for setting all the coordinates to 
1,1,1? or 0.1,0.1,0.1 if I still want it near the origin but biased toward the 
inside of the positive-going cell?
eab



On 12/14/2017 07:23 PM, James Holton wrote:

What I usually do for this is make a copy of the PDB file and change all the atom x-y-z positions to "1.000".  Then I use 
something like reforigin or my "origins.com 
<https://urldefense.proofpoint.com/v2/url?u=http-3A__origins.com=DwMGbg=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=kr4BAC6CGF8MkSeqVDxlDaQ3WprGVrjotZPIWrNrdts=RFDcNoPw6eYR8Aka4k3PWnf4_OIc98ZgWFd-LYwSoHo=>"
 script to shift the original coordinates via allowed symmetry operations, origin shifts, or perhaps indexing ambiguities until it is as 
close as possible to the "reference", which is at 1,1,1.  I use 1,1,1 instead of 0,0,0 because there are generally at least two 
symmetry-equivalent places that are equidistant from the origin. Declaring the reference to be a bit off-center breaks that ambiguity, and 
also biases the result toward having all-positive x,y,z values.


In case it is interesting, my script is here:

http://bl831.als.lbl.gov/~jamesh/scripts/origins.com 
<https://urldefense.proofpoint.com/v2/url?u=http-3A__bl831.als.lbl.gov_-7Ejamesh_scripts_origins.com=DwMGbg=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=kr4BAC6CGF8MkSeqVDxlDaQ3WprGVrjotZPIWrNrdts=9dlreI0nRWqq1Feor3gq_OOcpIxVPjpdRl2KTg9fMqg=>


You need to have the CCP4 suite set up for it to work.  Run it with no 
arguments to get instructions.


-James Holton

MAD Scientist


On 12/13/2017 5:50 AM, Kajander, Tommi A wrote:


Hello,

If someone could point this out would be very helpful... Wasnt there a 
simple script somewhere that would transfer coordinates close to origin - if 
they for some reason are not? Just cant find anything right away. Sure i have 
done this before...


Thanks,

Tommi

Re: [ccp4bb] coordinate transformation

[Edward A. Berry]

On 12/18/2017 05:39 AM, Eleanor Dodson wrote:

I showed you pdbset ..
Find the centre of mass for your assembly.
Move it where you will


Thanks. What James was suggesting was somewhat different (if I understood 
correctly): move EVERY ATOM to 1,1,1.
like:
  awk -v  FIELDWIDTHS="30 24 26" \
  '$1~/ATOM|HETATM/ && $2="   1.000   1.000   1.000" {print $1 $2 $3}' \
  3AEF.pdb
Then his origins program will find the symop/origin transform that moves the 
protein closest to that point. But come to think of it, moving COM to that 
point would probably have the same effect. Need to think about a lop-sided 
2-domain protein, where the symop is going to switch the orientation of 
large/small domains, and the large domain is negative of the com putting it 
outside the cell. but the com will be offset into the large domain, so probably 
still ok.

ORIGINS does assume like atoms have the same chain/resno in the different 
structures, which is not the case for 3AEF and friends, but they are close 
enough that it gives the right answer anyway.


pdbset xyzin mow.pdb
end
Find  CoM 0.7 1.3 -0.2

Hmm - a little thought - centre at 1 -1 0   say

pdbset yzin now.pdb xyzout changed.pdb

symgen x , y-2, z

end

New CoM  0.7 -0.7  -0.2

Eleanor





On 18 December 2017 at 00:19, Edward A. Berry <ber...@upstate.edu 
<mailto:ber...@upstate.edu>> wrote:

Neat idea!
And do you have a 1-line command for setting all the coordinates to 1,1,1? 
or 0.1,0.1,0.1 if I still want it near the origin but biased toward the inside 
of the positive-going cell?
eab


On 12/14/2017 07:23 PM, James Holton wrote:

What I usually do for this is make a copy of the PDB file and change all the atom x-y-z positions to 
"1.000".  Then I use something like reforigin or my "origins.com <http://origins.com>" 
script to shift the original coordinates via allowed symmetry operations, origin shifts, or perhaps indexing 
ambiguities until it is as close as possible to the "reference", which is at 1,1,1.  I use 1,1,1 instead 
of 0,0,0 because there are generally at least two symmetry-equivalent places that are equidistant from the origin. 
Declaring the reference to be a bit off-center breaks that ambiguity, and also biases the result toward having 
all-positive x,y,z values.


In case it is interesting, my script is here:

http://bl831.als.lbl.gov/~jamesh/scripts/origins.com 
<http://bl831.als.lbl.gov/~jamesh/scripts/origins.com>


You need to have the CCP4 suite set up for it to work.  Run it with no 
arguments to get instructions.


-James Holton

MAD Scientist


On 12/13/2017 5:50 AM, Kajander, Tommi A wrote:


Hello,

If someone could point this out would be very helpful... Wasnt 
there a simple script somewhere that would transfer coordinates close to origin 
- if they for some reason are not? Just cant find anything right away. Sure i 
have done this before...


Thanks,

Tommi

Re: [ccp4bb] coordinate transformation

[Edward A. Berry]

Neat idea!
And do you have a 1-line command for setting all the coordinates to 1,1,1? or 
0.1,0.1,0.1 if I still want it near the origin but biased toward the inside of 
the positive-going cell?
eab

On 12/14/2017 07:23 PM, James Holton wrote:

What I usually do for this is make a copy of the PDB file and change all the atom x-y-z positions to 
"1.000".  Then I use something like reforigin or my "origins.com" script to shift the 
original coordinates via allowed symmetry operations, origin shifts, or perhaps indexing ambiguities until it 
is as close as possible to the "reference", which is at 1,1,1.  I use 1,1,1 instead of 0,0,0 
because there are generally at least two symmetry-equivalent places that are equidistant from the origin. 
Declaring the reference to be a bit off-center breaks that ambiguity, and also biases the result toward 
having all-positive x,y,z values.


In case it is interesting, my script is here:

http://bl831.als.lbl.gov/~jamesh/scripts/origins.com


You need to have the CCP4 suite set up for it to work.  Run it with no 
arguments to get instructions.


-James Holton

MAD Scientist


On 12/13/2017 5:50 AM, Kajander, Tommi A wrote:


Hello,

If someone could point this out would be very helpful... Wasnt there a simple 
script somewhere that would transfer coordinates close to origin - if they for 
some reason are not? Just cant find anything right away. Sure i have done this 
before...


Thanks,

Tommi

Re: [ccp4bb] Crystal structure of an unknown protein

[Edward A. Berry]

In this case you know the protein is closely relaed to whatefer contaminer 
found, and you have good sequence data, so I agree the next step is blast. 
Maybe it is an isozyme of the structure used.

In cases where you solve an unknown by heavy atom derivatives and have no idea 
what it is; and the resolution is too low to get a good read on the sequence, 
but you can trace the chain; the DALI server is the way to go. That is how we 
identified 4HEI.

On 12/14/2017 07:08 AM, Jiyong Su wrote:

Dear CCP4bb,

In 2014, I collected a high quality data set from a crystal. But I could not 
solve the structure of that crystal because this protein is a contaminate.
Recently, I used StruBE's Contaminer and fortunately got the solution. Thanks 
ContaMiner!!!  This protein is a contaminate protein.

However, I found this protein is an unknown protein (about 180 residues) whose 
amino acid sequence is not totally same as E.coli. There are about 20 point 
mutation sites comparing to the E.coli protein. This means this protein may be 
from an unknown bacteria.

The space group of this crystal is new. There is also a new ligand in this 
protein.

My question is how could I found the primary structure of this protein and how 
to deposit this protein in PDB.

Best regards,

Jiyong

Re: [ccp4bb] coordinate transformation

[Edward A. Berry]

On 12/14/2017 04:34 AM, Tim Gruene wrote:

Dear Tommi,


1.

if you only need to consider translations, and not other symmetry operations,
you can use moleman2, convert coordinates to fractional ones and add or
substract the integer that brings the centre of mass closest to 0.


2.

In case you want to take the symmetry operations into account, you would have
to check for each operator, which one brings the centre of mass closest to 0.
This could most likely be scripted with moleman2.


3. If you also want to consider origin shifts (in spacegroups where alternate 
origins exist) the achesym site does that. (If you can't bring the molecule to 
the origin, bring the origin to the molecule)

4. If you also want to pack multiple chains that may have been built in various 
locations into a compact multimer, the achesym site does that.

But no, it's not a simple script.
And I believe the achesym site works not so much to put the center of mass near the 
origin, but to put as many atoms as possible in first unit cell (0 on behalf of Paul Emsley
 Sent: Thursday, December 14, 2017 3:25:19 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] coordinate transformation

On 13/12/2017 13:50, Kajander, Tommi A wrote:

Hello,

If someone could point this out would be very helpful... Wasnt there a
simple script somewhere that would transfer coordinates close to origin -
if they for some reason are not? Just cant find anything right away.

At the risk of not answering the question because it's not a simple script,
my I recommend Coot?

File -> Open -> yourcoords.cif
Draw -> Cell & Symm -> Master Switch -> Yes
Show Unit Cells -> Yes
OK
Drag the View to the Origin # it's marked with an "O"
Middle-mouse click on an Symmetry-related Atom # that's close to the origin
Extensions -> Modelling -> Symm Shift Reference Chain Here


--
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OFLC/104
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

Re: [ccp4bb] new ContaMiner features

[Edward A. Berry]

My 2 cents worth:
I think contaminer is an extremely useful service. I may be a sloppy biochemist,
but I am not the only one. There are multiple structures in the database of say
bacterioferritin or AcrB that were solved from crystals that were supposed to
be something else. I remember in a discussion with the organizer of my session
at a Gordon conference, she excitedly announced that there would be preliminary
crystallographic data on respiratory Complex I. But by the time of the 
conference
the authors discovered they had crystallized something else. And the beautiful 
crystals
of Paracoccus Complex II (from Doug Rees's lab?) that graced the catalog of
Hampton Research (And I believe were part of the basis for the first membrane
protein screen) never saw publication.  The authors of
  http://www.sciencedirect.com/science/article/pii/S0304416506000894
certainly feel there is a real problem.  Some proteins crystallize readily even 
when
present as minor contaminants. And some protein complexes become more 
heterogeneous
if over-purified due to partial loss of loosely-bound subunits.
Most of my career I've worked with high-abundance natural-source proteins.
During a recent foray into the realm of overexpressed proteins, my group has
crystallized (and solved) at least a half dozen wrong proteins from E. coli.
I spent months on one of these (ATCase in Rhomb sg with low-level 
obverse/reverse
twinning that caused it to sometimes index as P3) Then solved the rest rapidly
by checking the closest several hits with nearest-cell.  All of these E.coli 
proteins
were already present in the PDB. I wonder how many were from accidental 
crystals.
And now bacterioferritin (this time from M. smegmatis) keeps coming back to 
haunt us.

I would say any time with a new crystal when a molecular replacement 
unexpectedly fails,
and even before you start to collect heavy atom or selenomet data, it would be 
worth
to submit to nearest-cell and contaminer. I would be more likely to question the
utility of an anisotropy correction server, given that modern maximum-likelihood
refinement programs can deal with weak data satisfactorily (speaking from
ignorance- I'm sure supporting evidence and examples exist, I just haven't
bothered to look them up. And I know my colleagues here at Upstate have used
anisotropy correction to good effect with a difficult problem- I hope they
weren't using filled-in maps!)
eab

On 11/23/2017 03:24 PM, Tristan Croll wrote:

Dear Radu,

I think this is a little harsh. Biology is a fabulously messy thing, and very 
prone to doing the unexpected. See the excellent paper by Niedzialkowska et al. 
at https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4815408/#!po=13.6905 for some 
examples. Sometimes unexpected things (which just happen to have a similar size 
to your target) carry through all the purification steps - I remember having 
terrible trouble isolating his-tagged IGF-I (not for crystallization) from Sf9 
lysates due to a cathepsin-like protease that stuck doggedly to the Ni-NTA 
column even under 8M urea, yet co-eluted in imidazole. Even if contaminant 
proteins are barely visible on your SDS-PAGE gel, if they crystallise easily 
and your target doesn’t...  all these things and many others have happened, and 
have undoubtedly driven the occasional poor grad student to the brink of giving 
it all up.

I guess in these days of relatively cheap and ubiquitous mass spec it may make 
sense to sacrifice a crystal to trypsin digest and MS/MS sequencing just for 
peace of mind, but in the average case I think that’s likely to be overkill. 
Shooting crystals at a synchrotron is now very routine, so I think it makes 
perfect sense to provide a computational check for the (hopefully rare) 
surprise case.

Best regards,

Tristan
Tristan Croll
Research Fellow
Cambridge Institute for Medical Research
University of Cambridge CB2 0XY


On 23 Nov 2017, at 19:35, r...@mrc-lmb.cam.ac.uk 
 wrote:


Dear Stefan,

Just a couple of thoughts:

- first of all I think that Gerard is absolutely right, it would have been
nice to raise such issues first with the developers. In my experience,
Staraniso does a fantastic job if used correctly.

- but if you're OK with public trials, may I ask: why on Earth would anybody
need ContaMiner? Are you trying to offer some sort of computational cure for
sloppy biochemistry? There is zero point in crystallizing crap samples, sorry
to say this. In my 17 or so years in Strubi I've never heard of anybody
crystallizing a "contaminant", being it a purification tag or whatever.

I suppose this might have happened to somebody you know, hence the motivation
to spend time on the bizarre ContaMiner. Which is a pity, a silly outcome
would only teach people to do their job (or train their robots) properly.

Best wishes,

Radu

--
Radu Aricescu
MRC Laboratory of Molecular Biology
Francis Crick Avenue
Cambridge Biomedical Campus
Cambridge CB2 0QH, U.K.
tel: 

Re: [ccp4bb] Scripting for COOT

[Edward A. Berry]

(set-go-to-atom-molecule 0)
(set-go-to-atom-chain-residue-atom-name "B" 42 " CA ")


Would those be also on the command line, or where?

On 11/16/2017 07:19 AM, Paul Emsley wrote:

On 16/11/2017 08:08, Martín Martínez Ripoll wrote:


I am trying to write a long script that, among others, runs COOT, and for this 
purpose we use something like:

coot  --pdb  refmac-out.pdb--auto refmac-out.mtz

However, I do not know how to include in the script an instruction or keyword 
to centre at a particular
residue number.

Does anybody know how to do it?


Well, presuming that refmac-out.pdb creates a molecule with index 0, you could 
use

(set-go-to-atom-molecule 0)
(set-go-to-atom-chain-residue-atom-name "B" 42 " CA ")

Paul.

Re: [ccp4bb] model bias

[Edward A. Berry]

And given that all these maps (Fo, Fc, and their differences)
are made without the F000 term and so have average value zero,
it is highly likely that Fc will be negative in the region
of the deleted subunit (solvent is the lowest density in the model
except for the chinks of vacuum between the atoms in the protein,
and averaging over the volume of the protein you expect protein
to be electron-denser than solvent).
If Fc is negative then Fo-Fc negative implies Fo is still
more negative, and 2fo-fc cannot be positive.

It would be instructive to make Fo and Fc maps and check
their values in the region of the deleted subunit to resolve
the contradiction.


On 10/11/2017 04:47 PM, Edward A. Berry wrote:

This is hard to understand/impossible, at least in terms of
simple 2fo-fc and fo-fc maps.
We can think of a difference map as the difference between two maps,
like fo-fc map = fo map - fc map.
There is no model in the region, since you deleted it,
so Fc=0, and Fo-Fc negative => Fo<0, so 2fo-fc cannot be positive.

No, wait, there will be solvent, so Fc = Rho(solvent)
Fo-Fc is negative, so Fo <Rho(solvent)
2Fo-Fc positive => 2Fo>Rho(solv)
2Fo > Fc > Fo
Fo > 1/2 Fc
So this would be possible if the density of your protein is less than
that of the solvent but more than half that of the solvent.
Partial occupancy? but where the protein is missing there
would be solvent (in Fo if not in Fc),
so you still couldn't get below density of the solvent.
Maybe its a problem with the solvent model?

On 10/11/2017 09:48 AM, Karsten Dreifus wrote:

Dear all,
I have a 120 aa protein. Matthews coefficient indicates 3 mol in asu.
Molrep (template with 70 % seq identity)  finds three NCS molecules
(the template model has only 1 chain as it is in different space
group). Now, REFMAC refines it to 25/30 % R/RFREE. I was just worried
about model bias, so I just deleted chain C and re-ran the refinement.
I observe that in the place of chain C, the DELFWT map in Coot shows
negative density (red colour). Shouldnt the deleted regions show
POSITIVE density (green colour)? The 2fo-fc map in the deleted region
shows usual blue colour. I shook the 2chain model with Phenix simple
dynamics and then ran refinement with same results.

How do I verify the model is correct?
Karsten

Re: [ccp4bb] model bias

[Edward A. Berry]

This is hard to understand/impossible, at least in terms of
simple 2fo-fc and fo-fc maps.
We can think of a difference map as the difference between two maps,
like fo-fc map = fo map - fc map.
There is no model in the region, since you deleted it,
so Fc=0, and Fo-Fc negative => Fo<0, so 2fo-fc cannot be positive.

No, wait, there will be solvent, so Fc = Rho(solvent)
Fo-Fc is negative, so Fo  2Fo>Rho(solv)
2Fo > Fc > Fo
Fo > 1/2 Fc
So this would be possible if the density of your protein is less than
that of the solvent but more than half that of the solvent.
Partial occupancy? but where the protein is missing there
would be solvent (in Fo if not in Fc),
so you still couldn't get below density of the solvent.
Maybe its a problem with the solvent model?

On 10/11/2017 09:48 AM, Karsten Dreifus wrote:

Dear all,
I have a 120 aa protein. Matthews coefficient indicates 3 mol in asu.
Molrep (template with 70 % seq identity)  finds three NCS molecules
(the template model has only 1 chain as it is in different space
group). Now, REFMAC refines it to 25/30 % R/RFREE. I was just worried
about model bias, so I just deleted chain C and re-ran the refinement.
I observe that in the place of chain C, the DELFWT map in Coot shows
negative density (red colour). Shouldnt the deleted regions show
POSITIVE density (green colour)? The 2fo-fc map in the deleted region
shows usual blue colour. I shook the 2chain model with Phenix simple
dynamics and then ran refinement with same results.

How do I verify the model is correct?
Karsten

Re: [ccp4bb] ccp4 website not secure

[Edward A. Berry]

OK, on second thought it might not be a good idea to downgrade the browser:

On 10/11/2017 12:49 PM, Guillaume Gaullier wrote:

Hello Edward,

This website has some justification for using HTTPS everywhere: 
https://https.cio.gov/everything

Downgrading a web browser is probably the worst advice you can possibly give to 
someone.
You might be interested in this Q thread explaining why: 
https://security.stackexchange.com/questions/67596/why-is-it-a-security-problem-not-to-update-ones-browser

I hope you will consider amending your last message, to prevent people 
subscribed to ccp4bb from exposing themselves with out of date browsers.

With best wishes,

Guillaume



On Oct 11, 2017, at 10:34, Edward A. Berry <ber...@upstate.edu> wrote:

Is it because of this? Its been coming for a while now:
https://blog.mozilla.org/security/2015/04/30/deprecating-non-secure-http/

I see no reason why a website, dedicated to providing information
available to everyone, should be required to use https.
The web is too focused on e-commerce, where security is more important.

I hope firefox can be configured to allow insecure http, perhaps with a warning.
Otherwise downgrade to previous version (FF 47 connects fine).
Just be sure when visiting ccp4.ac.uk, don't enter personal info
like your tax-payer ID or credit card number. If you need to login
to modify the wiki or your subscription details, use another password for
the bank.

eab

On 10/11/2017 05:15 AM, Markus Heckmann wrote:

If anyone from CCP4 website has noticed that...
Your connection is not secure


The owner of www.ccp4.ac.uk has configured their web site improperly.
To protect your information from being stolen, Firefox has not
connected to this web site.


(https warning message)
https://www.ccp4.ac.uk/ccp4online/

Re: [ccp4bb] ccp4 website not secure

[Edward A. Berry]

Is it because of this? Its been coming for a while now:
https://blog.mozilla.org/security/2015/04/30/deprecating-non-secure-http/

I see no reason why a website, dedicated to providing information
available to everyone, should be required to use https.
The web is too focused on e-commerce, where security is more important.

I hope firefox can be configured to allow insecure http, perhaps with a warning.
Otherwise downgrade to previous version (FF 47 connects fine).
Just be sure when visiting ccp4.ac.uk, don't enter personal info
like your tax-payer ID or credit card number. If you need to login
to modify the wiki or your subscription details, use another password for
the bank.

eab

On 10/11/2017 05:15 AM, Markus Heckmann wrote:

If anyone from CCP4 website has noticed that...
Your connection is not secure


The owner of www.ccp4.ac.uk has configured their web site improperly.
To protect your information from being stolen, Firefox has not
connected to this web site.


(https warning message)
https://www.ccp4.ac.uk/ccp4online/

Re: [ccp4bb] HKL to mtz

[Edward A. Berry]

On 10/05/2017 11:01 PM, ameya benz wrote:

Hi,

I want to convert HKL file to mtz. I tried using F2mtz but somehow the output 
mtz is not working. What parameters should I set during conversion. Or can 
anyone suggest alternative to F2mtz?

regards,
Ameya
National chemical laboratory, Pune, India


Hi, Ameya,
F2mtz works.
In order for someone to help you you need to give more information.
What does your HKL file look like (show us the first 10-20 lines)?
Are you running f2mtz from one of the GUIs, or from a a script or commandline?
What parameters did you give (or what was your script)? (often the FORMAT 
statement is the problem).
And in what way the output mtz file is not working? maybe use mtzdump to show 
what you got.
Hope that will lead to a speedy resolution!

Re: [ccp4bb] RMSD between superposed structures without moving

[Edward A. Berry]

Look at CCP4 "compar"
By default it averages RMSD over all atoms in mainchain and sidechain
of the residue, but if you first awk out only CA (or use pdbset to pick CA)
foreach structure, the mainchain value will presumably be the
Euclidian CA distance (This may require identical sequences,
at least no insertions):

#test with all-atom pdb files:
  cd $CEXAM/unix/runnable/
  ./refmac5_tls.exam
  ./compar.exam
output:
 RMS xyz and B AVERAGES RES , MAIN CH,SIDECHAIN
 RESMAIN CHAIN SIDE CHAIN MAIN CHAIN SIDE CHAINMAIN CHAIN 
SIDE CHAIN MAIN CHAIN SIDE CHAIN
   1 ASP A
  0.21 10.10.44 24.9
   2 VAL A
  0.17 10.00.19 11.1
   3 SER A
  0.36 10.20.14  6.0
 . . .

#Awk the CA's into two new files:
   awk '$1~/ATOM/ && $3~/CA/' $CCP4_SCR/rnase_out.pdb > 
$CCP4_SCR/rnase_outCA.pdb
   awk '$1~/ATOM/ && $3~/CA/' $CEXAM/rnase/rnase.pdb > $CCP4_SCR/rn

compar  \
  XYZIN1 $CCP4_SCR/rnaseCA.pdb \
  XYZIN2 $CCP4_SCR/rnase_outCA.pdb \
  RMSTAB $CCP4_SCR/rnaseCA.rms\
  << END-compar
 comparing rnase coordinates before and after refinement
 2
 3.0 16
 END-compar

 RMS xyz and B AVERAGES RES , MAIN CH,SIDECHAIN
 RESMAIN CHAIN SIDE CHAIN MAIN CHAIN SIDE CHAINMAIN CHAIN 
SIDE CHAIN MAIN CHAIN SIDE CHAIN
   1 ASP A
  0.24 13.30.00  0.0
   2 VAL A
  0.12  6.60.00  0.0
   3 SER A
  0.05  8.10.00  0.0
   4 GLY A
  0.29  6.40.00  0.0
   5 THR A
  0.11  8.50.00  0.0
   6 VAL A
. . .

#compare with independently calculated distances:
pdbd2b $CCP4_SCR/rnaseCA.pdb $CCP4_SCR/rnase_outCA.pdb 1 0 | moreFind distances 
greater than threshold between corresponding atoms in 2 PDB files
Usage: pdbd2b file1 file2 startres# [thresh]
  CA  ASP A   1  CA  ASP A   1  0.2376
  CA  VAL A   2  CA  VAL A   2  0.1173
  CA  SER A   3  CA  SER A   3  0.0462
  CA  GLY A   4  CA  GLY A   4  0.2865
  CA  THR A   5  CA  THR A   5  0.1128
  CA  VAL A   6  CA  VAL A   6  0.2899


On 08/27/2017 07:09 AM, Johannes Sommerkamp wrote:

Hello everybody,
I have superposed two structures based on the central beta-sheet CA atoms with the 
"super" command in Pymol.
Now, I want to calculate the RMSD between ALL atoms or ALL CA atoms without moving the 
structures again. The rms_cur command in Pymol would do that, but only works if all atom 
identifiers match. Adding "transform=0" to the super, oder align command still 
does the alignment and moves the structure but does not show the movement.

Is there an easy way to just calculate the all atom RMSD between two already 
superposed structures in pymol or any other programm?

Thanks in advance!
Johannes

Re: [ccp4bb] weird diffraction pattern

[Edward A. Berry]

Thanks for spelling it out!
Would that advice still hold if the mosaicity of the crystal is 0.7 degrees?
(I know, I should go read the paper., but . . .)
eab

On 07/13/2017 03:00 PM, Gerard Bricogne wrote:

Dear Gerd,

  I can assure you that I have no shares in Dectris nor any
commecial connections with them. What I do have is a lot of still
vivid memories of CCD images, with their wooly point-spread function
that was affected by fine-grained spatial variability as well as by
irredicible inaccuracies in the geometric corrections required to try
and undo the distortions introduced by the fiber-optic taper. By
comparison the pixel-array detectors have a very regular structure, so
that slight deviations from exact registering of the modules can be
calibrated with high accuracy, making it possible to get very small
residuals between calculated and observed spot positions. That, I
certainly never saw with CCD images.

  I do think that asking for the image width was a highly pertinent
question in this case, that had not been asked. As a specialist you
might know how to use a CCD to good effect in fine-slicing mode, but
it is amazing how many people there are still out there who are told
to use 0.5 or even 1.0 degree image widths.

  Compensating the poor PSF of a CCD by fine slicing in the angular
dimension is a tall order. With a Pilatus at 350mm from the crystal,
the angular separation between 174-micron pixels is 0.5 milliradian.
To achieve that separation in the angular (rotation) dimension, the
equivalent image width would have to be 0.03 degree. For an EIGER the
numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree.

  Hence my advice, untainted by any commercial agenda :-) .


  With best wishes,

   Gerard.

--
On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote:

Dear Gerard,

you sound like a sales person for Dectris. Fine slicing is perfectly fine
with CCD detectors - it takes a bit longer because of the step scan instead
of continuous scan. The read noise issue is often overstated compared to the
sample induced scatter background. If for fine slicing at 0.05 degree or
less the diffraction peaks go too close to the read noise make a longer
exposure - signal goes up, ratio signal to sample-induced-BG less, as for
any fine slicing, same read noise.

It would be helpful to analyze the dense spot packing along layer lines if
we knew the wavelength and the sample-to-detector distance (assuming this is
a 300 mm detector) and the rotation width - as you pointed out. That would
help to distinguish between multiple crystals (my guess) and lattice
translocation disorder. Fine slicing is definitely needed to figure out what
the diffraction pattern at 120 degree could tell you in terms of strong
anisotropy .

Best regard.

Gerd

On 13.07.2017 08:20, Gerard Bricogne wrote:

Dear Tang,

  I noticed that your diffraction images seem to have been recorded
on a 3x3 CCD detector. With this type of detector, fine slicing is
often discouraged (because of the readout noise), and yet with the two
long cell axes you have, any form of thick (or only semi-fine) slicing
would result in spot overlaps.

  What, then, was your image width? Would you have access to a
beamline with a Pilatus detector so that you could collect fine-sliced
data?

  I would tend to agree with Herman that your crystals might be
cursed with lattice translocation disorder (LTD), but you might as
well try and put every chance of surviving this on your side by making
sure that you collect fine-sliced data. LTD plus thick slicing would
give you random data along the streaky direction. Use an image width
of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS
to process your images.


  Good luck!
Gerard

--
On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote:

Hi David,
Thanks for your comments. Although the spots become streaky in certain 
directions, I have processed the data in HKL3000 and imosflm, which suggested 
the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), 
completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with 
the solved native structure, the molecular replacement solution was poor. So I 
ran Balbes with the split domains of the native structure. Although the 
solutions were also poor, I found the MR score of one solution above 35. On the 
basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. 
Unfortunately, there are four molecules in the asymmetric unit and it is to 
hard for me to reduce the Rfree further.

All best,

Chenjun Tang

Re: [ccp4bb] Rmergicide Through Programming

[Edward A. Berry]

But R-merge is not really narrower as a fraction of the mean value- it just 
gets smaller proportionantly as all the numbers get smaller:
RMSD of .0043 for R-meas multiplied by factor of 0.022/.027 gives 0.0035 which 
is the RMSD for Rmerge. The same was true in the previous example. You could 
multiply R-meas by .5 or .2 and get a sharper distribution yet! And that factor 
would be constant, where this only applies for super-low redundancy.

On 07/08/2017 03:23 PM, James Holton wrote:


The expected distribution of Rmeas values is still wider than that of Rmerge 
for data with I/sigma=30 and average multiplicity=2.0. Graph attached.

I expect that anytime you incorporate more than one source of information you 
run the risk of a noisier statistic because every source of information can 
contain noise.  That is, Rmeas combines information about multiplicity with the 
absolute deviates in the data to form a statistic that is more accurate that 
Rmerge, but also (potentially) less precise.

Perhaps that is what we are debating here?  Which is better? accuracy or 
precision?  Personally, I prefer to know both.

-James Holton
MAD Scientist

On 7/8/2017 11:02 AM, Frank von Delft wrote:


It is quite easy to end up with low multiplicities in the low resolution shell, 
especially for low symmetry and fast-decaying crystals.

It is this scenario where Rmerge (lowres) is more misleading than Reas.

phx


On 08/07/2017 17:31, James Holton wrote:

What does Rmeas tell us that Rmerge doesn't?  Given that we know the 
multiplicity?

-James Holton
MAD Scientist

On 7/8/2017 9:15 AM, Frank von Delft wrote:


Anyway, back to reality:  does anybody still use R statistics to evaluate anything other than 
/strong/ data?  Certainly I never look at it except for the low-resolution bin (or strongest 
reflections). Specifically, a "2%-dataset" in that bin is probably healthy, while a 
"9%-dataset" probably Has Issues.

In which case, back to Jacob's question:  what does Rmerge tell us that Rmeas 
doesn't.

phx




On 08/07/2017 17:02, James Holton wrote:

Sorry for the confusion.  I was going for brevity!  And failed.

I know that the multiplicity correction is applied on a per-hkl basis in the 
calculation of Rmeas.  However, the average multiplicity over the whole 
calculation is most likely not an integer. Some hkls may be observed twice 
while others only once, or perhaps 3-4 times in the same scaling run.

Allow me to do the error propagation properly.  Consider the scenario:

Your outer resolution bin has a true I/sigma = 1.00 and average multiplicity of 2.0. 
Let's say there are 100 hkl indices in this bin.  I choose the "true" 
intensities of each hkl from an exponential (aka Wilson) distribution. Further assume the 
background is high, so the error in each observation after background subtraction may be 
taken from a Gaussian distribution. Let's further choose the per-hkl multiplicity from a 
Poisson distribution with expectation value 2.0, so 0 is possible, but the long-term 
average multiplicity is 2.0. For R calculation, when multiplicity of any given hkl is 
less than 2 it is skipped. What I end up with after 120,000 trials is a distribution of 
values for each R factor.  See attached graph.

What I hope is readily apparent is that the distribution of Rmerge values is taller and 
sharper than that of the Rmeas values.  The most likely Rmeas is 80% and that of Rmerge 
is 64.6%.  This is expected, of course.  But what I hope to impress upon you is that the 
most likely value is not generally the one that you will get! The distribution has a 
width.  Specifically, Rmeas could be as low as 40%, or as high as 209%, depending on the 
trial.  Half of the trial results falling between 71.4% and 90.3%, a range of 19 
percentage points.  Rmerge has a middle-half range from 57.6% to 72.9% (15.3 percentage 
points).  This range of possible values of Rmerge or Rmeas from data with the same 
intrinsic quality is what I mean when I say "numerical instability".  Each and 
every trial had the same true I/sigma and multiplicity, and yet the R factors I get vary 
depending on the trial.  Unfortunately for most of us with real data, you only ever get 
one trial, and you can't predict which
Rmeas or Rmerge you'll get.

My point here is that R statistics in general are not comparable from 
experiment to experiment when you are looking at data with low average 
intensity and low multiplicity, and it appears that Rmeas is less stable than 
Rmerge.  Not by much, mind you, but still jumps around more.

Hope that is clearer?

Note that in no way am I suggesting that low-multiplicity is the right way to 
collect data.  Far from it.  Especially with modern detectors that have 
negligible read-out noise. But when micro crystals only give off a handful of 
photons each before they die, low multiplicity might be all you have.

-James Holton
MAD Scientist



On 7/7/2017 2:33 PM, Edward A. 

Re: [ccp4bb] Rmergicide Through Programming

[Edward A. Berry]

I think the confusion here is that the "multiplicity correction" is applied
on each reflection, where it will be an integer 2 or greater (can't estimate
variance with only one measurement). You can only correct in an approximate
way using using the average multiplicity of the dataset, since it would depend
on the distribution of multiplicity over the reflections.

And the correction is for r-merge. You don't need to apply a correction
to R-meas.
R-meas is a redundancy-independent best estimate of the variance.
Whatever you would have used R-merge for (hopefully taking allowance
for the multiplicity) you can use R-meas and not worry about multiplicity.
Again, what information does R-merge provide that R-meas does not provide
in a more accurate way?

According to the denso manual, one way to artificially reduce
R-merge is to include reflections with only one measure (averaging
in a lot of zero's always helps bring an average down), and they say
there were actually some programs that did that. However I'm
quite sure none of the ones we rely on today do that.

On 07/07/2017 03:12 PM, Kay Diederichs wrote:

James,

I cannot follow you. "n approaches 1" can only mean n = 2 because n is integer. 
And for n=2 the sqrt(n/(n-1)) factor is well-defined. For n=1, neither contributions to 
Rmeas nor Rmerge nor to any other precision indicator can be calculated anyway, because 
there's nothing this measurement can be compared against.

just my 2 cents,

Kay

On Fri, 7 Jul 2017 10:57:17 -0700, James Holton  
wrote:


I happen to be one of those people who think Rmerge is a very useful
statistic.  Not as a method of evaluating the resolution limit, which is
mathematically ridiculous, but for a host of other important things,
like evaluating the performance of data collection equipment, and
evaluating the isomorphism of different crystals, to name a few.

I like Rmerge because it is a simple statistic that has a simple formula
and has not undergone any "corrections".  Corrections increase
complexity, and complexity opens the door to manipulation by the
desperate and/or misguided.  For example, overzealous outlier rejection
is a common way to abuse R factors, and it is far too often swept under
the rug, sometimes without the user even knowing about it.  This is
especially problematic when working in a regime where the statistic of
interest is unstable, and for R factors this is low intensity data.
Rejecting just the right "outliers" can make any R factor look a lot
better.  Why would Rmeas be any more unstable than Rmerge?  Look at the
formula. There is an "n-1" in the denominator, where n is the
multiplicity.  So, what happens when n approaches 1 ?  What happens when
n=1? This is not to say Rmerge is better than Rmeas. In fact, I believe
the latter is generally superior to the first, unless you are working
near n = 1. The sqrt(n/(n-1)) is trying to correct for bias in the R
statistic, but fighting one infinity with another infinity is a
dangerous game.

My point is that neither Rmerge nor Rmeas are easily interpreted without
knowing the multiplicity.  If you see Rmeas = 10% and the multiplicity
is 10, then you know what that means.  Same for Rmerge, since at n=10
both stats have nearly the same value.  But if you have Rmeas = 45% and
multiplicity = 1.05, what does that mean?  Rmeas will be only 33% if the
multiplicity is rounded up to 1.1. This is what I mean by "numerical
instability", the value of the R statistic itself becomes sensitive to
small amounts of noise, and behaves more and more like a random number
generator. And if you have Rmeas = 33% and no indication of
multiplicity, it is hard to know what is going on.  I personally am a
lot more comfortable seeing qualitative agreement between Rmerge and
Rmeas, because that means the numerical instability of the multiplicity
correction didn't mess anything up.

Of course, when the intensity is weak R statistics in general are not
useful.  Both Rmeas and Rmerge have the sum of all intensities in the
denominator, so when the bin-wide sum approaches zero you have another
infinity to contend with.  This one starts to rear its ugly head once
I/sigma drops below about 3, and this is why our ancestors always
applied a sigma cutoff before computing an R factor.  Our small-molecule
colleagues still do this!  They call it "R1".  And it is an excellent
indicator of the overall relative error.  The relative error in the
outermost bin is not meaningful, and strangely enough nobody ever
reported the outer-resolution Rmerge before 1995.

For weak signals, Correlation Coefficients are better, but for strong
signals CC pegs out at >95%, making it harder to see relative errors.
I/sigma is what we'd like to know, but the value of "sigma" is still
prone to manipulation by not just outlier rejection, but massaging the
so-called "error model".  Suffice it to say, crystallographic data
contain more than one type of error.  Some sources are important for
weak 

Re: [ccp4bb] RMSD between unaligned structures

[Edward A. Berry]

I second the use of LSQMAN.
The command for calculating wo aligning appears to be:
rmsd_calc (abbreviated rm).
   http://xray.bmc.uu.se/usf/lsqman_man.html#S59

If you have multiple chains, use the option "chain_mode original"
before loading the pdb's to avoid renaming chains:
   http://xray.bmc.uu.se/usf/lsqman_man.html#S25

On 07/03/2017 06:32 AM, Stéphane Duquerroy wrote:

Hi James
LSQMAN can calculate the current RMSD between the 2 models (RMsd_calc mol1 
range1 mol2 range2 [Ltarget])
Be careful it renames the chain names

Stephane

---
Duquerroy Stéphane
Structural Virology Unit - PASTEUR INSTITUTE
25 rue du Dr Roux, 75015 Paris, France
lab: +33 (0)1 45 68 82 66
fax: +33 (0)1 45 68 89 93
email: sduq...@pasteur.fr




--
*De: *"James Foadi" <09daa8ec3774-dmarc-requ...@jiscmail.ac.uk>
*À: *
*Envoyé: *Lundi 3 Juillet 2017 11:47:00
*Objet: *[ccp4bb] RMSD between unaligned structures

Dear ccp4 tribe,
this might have been asked before, but I haven't paid enough attention.

I'd like to measure the RMSD between two models after molecular replacement. I 
can force the two models to overlap as much as possible within the symmetry and 
origin-shift constraints (using CSYMMATCH). But I don't want the program that 
compute RMSD to align the two structures. Can you suggest what I should use? 
And, perhaps, what keywords I should adopt?

Many thanks, in advance.

James
Dr James Foadi PhD Diamond Light Source Ltd. Diamond House Harwell Science and 
Innovation Campus Didcot Oxfordshire OX11 0DE United Kingdom office email: 
james.fo...@diamond.ac.uk alternative email: j.fo...@imperial.ac.uk personal 
web page: http://www.jfoadi.me.uk

Re: [ccp4bb] preparing DL-Malic acid stock solution

[Edward A. Berry]

(di)Sodium D,L-malate is available from Sigma-Aldrich (# CDS023113) and
probably dissolves to give a 3M solution which is slightly alkaline.
If pK2 is 5.1, then an insignificant amt of malic acid should bring the
pH down to 7 (If you have to add a significant amount, just add more
water to dilute to a final conc of 3M Malate + Malic acid)


On 06/08/2017 12:32 PM, Diana Tomchick wrote:

As the pKa1 of malic acid is 3.4, it may be that the partially neutralized 
malic acid salt is less soluble than the acid or the fully neutralized salt.

The pKa2 of malic acid is 5.1.

Contact the people at Hampton Research

https://www.hamptonresearch.com/contact_us.aspx

and ask them. They sell it as a 3.0 M solution, pH neutralized to 7.0.

It is possible that you need to fully neutralize it before it turns clear, or 
that you may also need to gently heat it.

Diana

**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas 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 Jun 8, 2017, at 9:54 AM, Sebastiano Pasqualato > wrote:


Dear all,
we’ve recently having trouble preparing a 3 M stock solution of DL-Malic Acid, 
pH 7 (which we had, so it’s doable!).
When we reach pH 3 - 4 the solution turns milky white and does not goes back to 
a clear solution even when the pH is raised.
Does anybody have any advice on how to get a clear solution? Has anyone gone 
through the same?
Thanks in advance,
ciao,
Sebastiano


--
*Sebastiano Pasqualato, PhD*
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
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Re: [ccp4bb] Problems with an exonuclease

[Edward A. Berry]

On 06/07/2017 10:46 AM, Bonsor, Daniel wrote:

It will either be two things. DNA or residual Triton-X-100.


Actually Triton X-100 absorbs more at 280 than 260 - in fact the hydroxyphenyl 
in TX-100 looks remarkably like tyrosine in RNase A. I long ago gave up hope of 
resolving triton from protein and DNA by spectral curve-fitting!
eab

Re: [ccp4bb] Conserved water

[Edward A. Berry]

Easiest way is to line up molecule pairs or chain pairs in COOT and see if 
there are equivalent waters.


If the number of waters is too large to inspect manually,
save the superposed structures to disk, grep out the waters from each into a 
separate files,
and use a program like http://www.cytbc1.com/berry/for/pdbdist3w.for
to compare files and write atoms closer than a specified threshold into a new 
pdb file

That program only compares two files, so you would have to do all possible 
pairs to get waters conserved
in at least 2 structures, or compare each file with the survivors from previous 
to get waters
conserved in all.
PS- It compiles with f77 (g77). May need touch-up for fortran90 (gfortran).
eab

On 06/07/2017 02:59 AM, Eleanor Dodson wrote:

Many years ago I wrote code to label waters with a code related to the 
residue/atom  they were Hbonded to , so then you could check whether all OH TYR 
227 in each chain  had an associated water.. But it used non-standard water 
naming ..

Easiest way is to line up molecule pairs or chain pairs in COOT and see if 
there are equivalent waters.

Eleanor

On 7 June 2017 at 00:30, gerardo andres <130afa955101-dmarc-requ...@jiscmail.ac.uk 
> wrote:

Hi everyone, does anyone know any strategy or program (besides pywater) to 
identify conserved waters in a protein?

Thanks,

Gerardo

Re: [ccp4bb] Off-topic: 3D printing tools for molecular models

[Edward A. Berry]

That is beautiful!

The pins-and-holes approach might be useful also for space-filling models
of multi-subunit complexes- both for holding the subunits together, and in
cases where one subunit partially encircles another, the subunit would be sliced
in half and pins could hold the halves together. That would make an educational
toy like a chinese puzzle, that you dissassemble and reassemble to see how the
subunits fit together. Something like cytochrome oxdase (13 subunits),
cytochrome bc1 (dimer of 11 subunits each),or Complex 1 (48? subunits).
eab

On 05/20/2017 09:37 AM, Paul Paukstelis wrote:

Sorry for the somewhat off-topic post.

After getting interested in 3D printing I quickly found that making nice
ball-and-stick objects was very difficult to do on the typical fused
filament printers most people can afford. This is largely because of the
amount of support structures needed for complicated objects.

I've been working on a Blender addon that takes VRML output from most
common programs (PyMol, Chimera, etc.) and allows the user to split the
model up to be printed as individual objects. It generates "pins" and
"holes" to allow the objects to be assembled post-printing.

It has gotten functional enough that I thought I would leave the link
here for those that are interested:

https://github.com/paukstelis/MolPrint

I have used it to print some pretty complex models!

https://thingiverse-production-new.s3.amazonaws.com/renders/0e/87/27/d8/0d/7f0cacf367cc2ed4b01c77705ff16767_preview_featured.JPG

Re: [ccp4bb] Revise Your Structure Without Changing the PDB Accession Code and Related Changes to the FTP Archive

[Edward A. Berry]

Couple of wuestions:
What is the procedure for updating an entry? Start a new submission, or mail 
revised coordinates along with an explanation of changes to deposit@rcsb?

"unchanged experimental data" - does this mean the exact same structure 
factors, or will newly reduced data from the same original diffraction images be 
acceptable?

Thanks,
Ed

On 05/17/2017 09:28 AM, Jasmine Young wrote:

*Use CAUTION opening attachments or clicking on links in emails - IMT Help 
Desk, 4-4115*



The wwPDB is planning to introduce in 2017 a new procedure for the management 
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At present, revised atomic coordinates for an existing released PDB entry are 
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The wwPDB is introducing a file versioning system that allows Depositors of 
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===

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

Actually this is taken care of in the BIOMOLECULE definition.
If the artist had used the principle biomolecule, it excludes the Fv fragments.

On 05/09/2017 01:08 PM, Edward A. Berry wrote:

In line with this, there are a number of pictures in the literature of the 
mitochondrial
electron transport chain, with the complexes lined up  in a row embedded in a 
membrane,
and with yeast complex III still having the Fv fragments it was crystallized 
with, attached.
Only obvious if you are familiar with the shape of the native structure, I 
guess.
Could have been avoided if the crystallographer had removed the Fv fragments 
before depositing,
but that would have been bad for the R-factors.

On 05/09/2017 12:49 PM, Ian Tickle wrote:


Hi Tristan

I'm not so sure.  The co-ordinates are the result of the experiment.  How other 
people choose to interpret those results is their affair.  Taking it to its 
logical conclusion suppose that we 'damage' the protein by mutating/deleting 
some residues or adding tags purely for the purpose of getting it to 
crystallise, do we report the structure of the protein as it is in the crystal, 
or do we report what it would have been if we hadn't messed with it ?  The 
choice is clear in that situation.

Cheers

-- Ian

On 9 May 2017 at 16:45, Tristan Croll <ti...@cam.ac.uk 
<mailto:ti...@cam.ac.uk>> wrote:

Hmm... this is a bit of a philosophical pickle in my mind. Do we want to 
model the structure as what it looks like after radiation damage has had its 
way with it, or what it must have looked like *before* the damage? I can see 
arguments both ways (and can sympathise with the former if you want to make 
radiation damage a subject of your manuscript), but this is going to lead to 
headaches for people who want to make use of the resulting coordinates to study 
the actual biology of your protein. Personally, I'd strongly prefer the latter 
approach.

Tristan


On 2017-05-09 16:06, Edward A. Berry wrote:

On 05/09/2017 06:18 AM, Ian Tickle wrote:

We have seen almost identical density to Ed's for GLU side-chains, with what looks like a linear molecule 
(yes exactly the size of CO2!) where the carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably making the same H bonds that the 
carboxylate was making to hold it there.  It would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid <https://en.wikipedia.org/wiki/Carbonic_acid> : "The hydration 
<https://en.wikipedia.org/wiki/Hydrate <https://en.wikipedia.org/wiki/Hydrate>> equilibrium constant 
<https://en.wikipedia.org/wiki/Equilibrium_constant <https://en.wikipedia.org/wiki/Equilibrium_constant>> 
at 25 °C is called K_h , which in the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 1.7×10^−3 in pure water^[5] 
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-HS-5
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-HS-5>> and ≈ 1.2×10^−3 in seawater 
<https://en.wikipedia.org/wiki/Seawater <https://en.wikipedia.org/wiki/Seawater>>.^[6] 
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-SB-6 
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-SB-6>> Hence, the majority of the carbon dioxide 
is not converted into carbo

n
ic

acid, remaining as CO_2 molecules.".


It looks like this ignores subsequent ionization of H2CO3 which would
be quite spontaneous at neutral pH.  However the Wikipedia article
also indicates the equilibrium is quite slow (which makes sense-
otherwise why would carbonic anhydrase exist?) and it would be a great
deal slower in vitreous ice at 100 K. Anyway, I had reached the same
conclusion and have modeled a number of the troublesome glutamates as
decarboxylated with CO2 hovering above. There is a problem that the
remaining CG tends to push the CO2 a little out of the density in some
cases, but not a severe clash and it may work itself out with further
refinement or manual assistance.
eab

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

In line with this, there are a number of pictures in the literature of the 
mitochondrial
electron transport chain, with the complexes lined up  in a row embedded in a 
membrane,
and with yeast complex III still having the Fv fragments it was crystallized 
with, attached.
Only obvious if you are familiar with the shape of the native structure, I 
guess.
Could have been avoided if the crystallographer had removed the Fv fragments 
before depositing,
but that would have been bad for the R-factors.

On 05/09/2017 12:49 PM, Ian Tickle wrote:


Hi Tristan

I'm not so sure.  The co-ordinates are the result of the experiment.  How other 
people choose to interpret those results is their affair.  Taking it to its 
logical conclusion suppose that we 'damage' the protein by mutating/deleting 
some residues or adding tags purely for the purpose of getting it to 
crystallise, do we report the structure of the protein as it is in the crystal, 
or do we report what it would have been if we hadn't messed with it ?  The 
choice is clear in that situation.

Cheers

-- Ian

On 9 May 2017 at 16:45, Tristan Croll <ti...@cam.ac.uk 
<mailto:ti...@cam.ac.uk>> wrote:

Hmm... this is a bit of a philosophical pickle in my mind. Do we want to 
model the structure as what it looks like after radiation damage has had its 
way with it, or what it must have looked like *before* the damage? I can see 
arguments both ways (and can sympathise with the former if you want to make 
radiation damage a subject of your manuscript), but this is going to lead to 
headaches for people who want to make use of the resulting coordinates to study 
the actual biology of your protein. Personally, I'd strongly prefer the latter 
approach.

Tristan


On 2017-05-09 16:06, Edward A. Berry wrote:

On 05/09/2017 06:18 AM, Ian Tickle wrote:

We have seen almost identical density to Ed's for GLU side-chains, with what looks like a linear molecule 
(yes exactly the size of CO2!) where the carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably making the same H bonds that the 
carboxylate was making to hold it there.  It would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid <https://en.wikipedia.org/wiki/Carbonic_acid> : "The hydration 
<https://en.wikipedia.org/wiki/Hydrate <https://en.wikipedia.org/wiki/Hydrate>> equilibrium constant 
<https://en.wikipedia.org/wiki/Equilibrium_constant <https://en.wikipedia.org/wiki/Equilibrium_constant>> 
at 25 °C is called K_h , which in the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 1.7×10^−3 in pure water^[5] 
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-HS-5
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-HS-5>> and ≈ 1.2×10^−3 in seawater 
<https://en.wikipedia.org/wiki/Seawater <https://en.wikipedia.org/wiki/Seawater>>.^[6] 
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-SB-6 
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-SB-6>> Hence, the majority of the carbon dioxide 
is not converted into carbo

n
ic

acid, remaining as CO_2 molecules.".


It looks like this ignores subsequent ionization of H2CO3 which would
be quite spontaneous at neutral pH.  However the Wikipedia article
also indicates the equilibrium is quite slow (which makes sense-
otherwise why would carbonic anhydrase exist?) and it would be a great
deal slower in vitreous ice at 100 K. Anyway, I had reached the same
conclusion and have modeled a number of the troublesome glutamates as
decarboxylated with CO2 hovering above. There is a problem that the
remaining CG tends to push the CO2 a little out of the density in some
cases, but not a severe clash and it may work itself out with further
refinement or manual assistance.
eab

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

Hmm... this is a bit of a philosophical pickle in my mind.


I agree.
Right now I want as accurate a model as possible to improve the phases for 
interpretation of a few remaining bits. I haven't decided what to deposit- 
maybe three separate structures:
1. Conservatively modeled: Everything that I can't model is left unmodelled.
2. Speculative: every little green blob is filled with partial-occupancy water, 
methanol, ethanol, acetate/bicarbonate, isopropanol, glycerol, Tris, or PEG 
fragments. If peroxy-glutamate fits better, put it.
3. repaired model- rebuild the damaged glutamates, cycteines and methionines. Average the two 
heterotetramers in the asymmetric unit to make one BioMolecule, and do a few ps of molecular 
dynamics to eliminate crystallization artifacts. (Since this would now be a "solution 
structure" I wouldn't be expected to report R-factor or deposit diffraction data for this 
one). More likely it would be rejected as a "Model".

eab

On 05/09/2017 11:45 AM, Tristan Croll wrote:

Hmm... this is a bit of a philosophical pickle in my mind. Do we want to model 
the structure as what it looks like after radiation damage has had its way with 
it, or what it must have looked like *before* the damage? I can see arguments 
both ways (and can sympathise with the former if you want to make radiation 
damage a subject of your manuscript), but this is going to lead to headaches 
for people who want to make use of the resulting coordinates to study the 
actual biology of your protein. Personally, I'd strongly prefer the latter 
approach.

Tristan

On 2017-05-09 16:06, Edward A. Berry wrote:

On 05/09/2017 06:18 AM, Ian Tickle wrote:

We have seen almost identical density to Ed's for GLU side-chains, with what looks like a linear molecule (yes 
exactly the size of CO2!) where the carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably making the same H bonds that the 
carboxylate was making to hold it there.  It would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid : "The hydration <https://en.wikipedia.org/wiki/Hydrate> 
equilibrium constant <https://en.wikipedia.org/wiki/Equilibrium_constant> at 25 °C is called K_h , which in 
the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 1.7×10^−3 in pure water^[5] 
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-HS-5> and ≈ 1.2×10^−3 in seawater 
<https://en.wikipedia.org/wiki/Seawater>.^[6] 
<https://en.wikipedia.org/wiki/Carbonic_acid#cite_note-SB-6> Hence, the majority of the carbon dioxide is 
not converted into car

b
o

n
ic

acid, remaining as CO_2 molecules.".


It looks like this ignores subsequent ionization of H2CO3 which would
be quite spontaneous at neutral pH.  However the Wikipedia article
also indicates the equilibrium is quite slow (which makes sense-
otherwise why would carbonic anhydrase exist?) and it would be a great
deal slower in vitreous ice at 100 K. Anyway, I had reached the same
conclusion and have modeled a number of the troublesome glutamates as
decarboxylated with CO2 hovering above. There is a problem that the
remaining CG tends to push the CO2 a little out of the density in some
cases, but not a severe clash and it may work itself out with further
refinement or manual assistance.
eab

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

On 05/09/2017 06:18 AM, Ian Tickle wrote:

We have seen almost identical density to Ed's for GLU side-chains, with what looks like a linear molecule (yes 
exactly the size of CO2!) where the carboxylate group would be and absolutely no density for the CG-CD bond.  So 
it's indeed very tempting to say that the CO2 is still there, and presumably making the same H bonds that the 
carboxylate was making to hold it there.  It would not be hydrated to carbonic acid, according to 
https://en.wikipedia.org/wiki/Carbonic_acid : "The hydration  
equilibrium constant  at 25 °C is called K_h , which in 
the case of carbonic acid is [H_2 CO_3 ]/[CO_2 ] ≈ 1.7×10^−3 in pure water^[5] 
 and ≈ 1.2×10^−3 in seawater 
.^[6] 
 Hence, the majority of the carbon dioxide is 
not converted into carbo

n
ic

acid, remaining as CO_2 molecules.".


It looks like this ignores subsequent ionization of H2CO3 which would be quite 
spontaneous at neutral pH.  However the Wikipedia article also indicates the 
equilibrium is quite slow (which makes sense- otherwise why would carbonic 
anhydrase exist?) and it would be a great deal slower in vitreous ice at 100 K. 
Anyway, I had reached the same conclusion and have modeled a number of the 
troublesome glutamates as decarboxylated with CO2 hovering above. There is a 
problem that the remaining CG tends to push the CO2 a little out of the density 
in some cases, but not a severe clash and it may work itself out with further 
refinement or manual assistance.
eab

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

As you suggest, it depends on the contour level. Looking through a list of 17 
dicarboxylates that I found problematic, there were 4 (actually two and their 
ncs-mates) that showed disconnected density for the carboxylate at 1.4 sigma as 
in the figure I sent. Going up to 2 sigma, four more became disconnected and 
linear (backbone density is continuous to 3.5 sigma). Going down to 0.6 sigma, 
disconnected residual density showed up for several more. So I guess there are 
varying degrees of decarboxylation, and varying extent of retention of the 
fragment.  I have the impression these are mostly on the protein/solvent 
boundary, which could explain their disorder, but perhaps would also expose 
them to greater concentration of radicals generated in the solvent channels(?).

Ed

On 05/04/2017 06:25 AM, Andrew Leslie wrote:

Dear Ed,

   I find your electron density quite interesting, because 
generally (I think, I would be happy to be corrected on this) when 
de-carboxylation of Asp/Glu occurs due to radiation damage, there is no 
evidence of what happens to the resulting CO2 group. One interpretation of this 
is that it diffuses away from the side chain and is effectively totally 
disordered, so no electron density is seen, but I was surprised that this would 
always be the case, especially as I would have thought that diffusion would be 
quite limited at 100K (maybe I’m wrong about that too, but that is supposed to 
be one reason why radiation damage is less at 100K).

If the residual density is due to partial de-carboxylation, then I would have 
expected density for the CG-CD bond, which is not present (at your chosen 
contour level).

Do many of your Glu side chains have the residual density?

Best wishes,

Andrew



On 3 May 2017, at 22:19, Edward A. Berry <ber...@upstate.edu> wrote:



On 05/03/2017 02:46 PM, Gerard Bricogne wrote:

Dear Ed,

  Have you considered the possibility that it could be a water
stepping in to fill the void created by partial decarboxylation of the
glutamate? That could be easily modelled, refined, and tested for its
ability to flatten the difference map.

  Gerard.


Actually some of them do appear decarboxylated. Is that something that can 
happen? In the crystal, or as radiation damage?
However when there is density for the carboxylate (figure), it appears 
continuous and linear, doesn't break up into spheres at H-bonding distance - 
almost like the CO2 is still sitting there- but I guess it would get hydrated 
to bicarbonate. I could use azide. Or maybe waters with some disorder.
Thanks,
eab

Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
comparison, not part of the model.

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

On 05/03/2017 02:46 PM, Gerard Bricogne wrote:

Dear Ed,

  Have you considered the possibility that it could be a water
stepping in to fill the void created by partial decarboxylation of the
glutamate? That could be easily modelled, refined, and tested for its
ability to flatten the difference map.

  Gerard.


Actually some of them do appear decarboxylated. Is that something that can 
happen? In the crystal, or as radiation damage?
However when there is density for the carboxylate (figure), it appears 
continuous and linear, doesn't break up into spheres at H-bonding distance - 
almost like the CO2 is still sitting there- but I guess it would get hydrated 
to bicarbonate. I could use azide. Or maybe waters with some disorder.
Thanks,
eab

Figure- 2mFo-DFc at 1.3 sigma, mFo-DFc at 3 sigma, green CO2 is shown for 
comparison, not part of the model.

Re: [ccp4bb] peroxy-glutamate?

[Edward A. Berry]

Thanks to all who replied. Yes, in some of the other cases density between Cd 
and Cg is conspicuously weak. From preliminary tests it looks like dual 
conformations, in some cases together with correlated waters, will account for 
the density adequately. No Zebras here!
eab

On 05/03/2017 04:26 AM, Matthew Merski wrote:

Have you tried just a double conformation of the Glu?  Its hard to tell in your 
pictures but it looks like there might be less than perfect density for the 
CG-CD bond?  If you just try adding a second conf of the Glu that might work 
too (and perhaps be a horse rather than a zebra making your hoofprints?)

Matthew Merski
Crystallochemistry Laboratory
Univ. of Warsaw

On Wed, May 3, 2017 at 6:29 AM, Edward A. Berry <ber...@upstate.edu 
<mailto:ber...@upstate.edu>> wrote:

I'm finishing up refinement of a 1.8A structure (R's 0.17, 0.20) , and among 
the largest peaks in the difference map are small spherical blobs that seem to be 
attached (1.46 A here) to carboxylate O's (Figures). Are these likely artifacts? 
If not, how can I interpret/model them? One idea is that the acid has reacted with 
peroxide from the PEG to make the (hydro)peroxy-acid. I don't know how stable that 
would be, and I don't see any peroxyglutamate in Ligand Depot or HIC-Up. Another 
guess would be acid hydroxamate but I don't know how that would be generated. 
Methyl ester seems to be ruled out by the proximity of the two water molecules 
(2.45 and 2.48 A here) suggesting the mystery atom is an H-bond acceptor or donor. 
However since the occupancy seems to be < 1, the waters may be there only when 
the atom is not.
I guess another possibility is there is a lot of motion in the plane of the 
carboxylate (up and down here) which cannot be modeled by my isotropic 
B-factors. In some cases the green blobs appear on both sides of the 
carboxylate (but that could also be alternate conformations of peroxyglutamate).

The difference map (mFo-DFc, green) is contoured at 3 sigma (.06 e-/A^3). 
The difference peak is 5.4 sigma (0.1 e/A3).
The 2mFo-DFc map is contoured at 1.5 sigma (0.1 e/A3). 2mFo-DFc density 
extends to the difference peak if I contour down at 0.64 sigma (0.04 e/A3, 
third figure).

If I put an O atom there, link it with plenty of slack, and refine 
occupancy, it goes to 1.54 A from the carboxylate O and refines to occupancy 
0.35, B-factor 15 (carboxylate O is 30). Now it is reached by 2mFo-DFc density 
at 1.5 sigma (0.1 e/A3).
Any suggestions would be welcome.
eab

Re: [ccp4bb] E.coli strain, knock-out for SDH/FR

[Edward A. Berry]

People who would know (and know whether such a strain would even be viable)
are Gary Cecchini at UC San Francisco/VA and Bob Gennis at Univ of Illinois
at Urbana. In case you are not already talking with them.

It has been reported that an assembly factor (SDH5 or SDHE or SDHAF2)
is required for flavination of both proteins, without which you would have
no activity. So knocking out that one gene may be sufficient. It has
been reported to be a little leaky however, perhaps more so at
higher temperature.  Meaning that SDH5-independent flavination has
been seen in some cases. And fumarate reductase has activity with
non-covalently bound FAD, but only for fumarate reduction not succinate
oxidation, so if "any kind of Complex II activity" only includes the
forward reaction, you would be OK.
There's a group in New Zealand working n SDHE in bacteria- pubmed SDHE
and see what is their latest, and they may have the knockout (of SDHE).
eab.

On 04/04/2017 10:54 AM, Fulvio Saccoccia, Sapienza wrote:

Dear ccp4ers,

I was wondering if someone does know (and hence could let me know in
turn) an E.coli strain which lacks both  of the sdh (succinate
dehydrogenase) and frd (fumarate reductase) operon, so that bacteria do
no longer retain any activity from complex II or similar. I know the
question is off-topic but I am confident about your kindness.

Cheers

Fulvio


--
Fulvio Saccoccia, PhD
Institute of Cell Biology and Neurobiology (IBCN)
Consiglio Nazionale delle Ricerche
Via E. Ramarini, 32 - 00015 Monterotondo Scalo Roma (RM)
tel.+390690091244

Re: [ccp4bb] protein precipitation reg

[Edward A. Berry]

On 03/30/2017 08:10 AM, mesters wrote:

If the pI of the protein is below the pH of the buffer (net negatively charged 
protein), optimum stabilization (salting out; lower solubility) of the 
macromolecule is achieved by combining a kosmotropic anion with a chaotropic 
cation, e.g. Ammoniumsulfate (most successful salt)!


??
According to the wikipedia page on Hoffmeister series, NH4+ is one of the 
_least_ chaotropic cations.


/For your pI 9.7 protein: Vice versa/, if the pI of the protein is above the pH 
of the buffer (net positively charged protein and thus inversion of the 
Hofmeister series), 50-150 mM Ammoniumsulfate is a far better choice for 
solubilisation than NaCl.//



That would explain why it is so hard to precipitate cytochrome c with NH4SO4!


/For your pI 5.6 protein:/Maybe you need a stronger "solubilizer" salt such as 
Nitrate or Thiocyanate while increasing the pH to 8.0 or 8.5 (to increase the net charge 
of the protein).

Good luck,

Jeroen


Am 29.03.17 um 15:38 schrieb Akilandeswari Gopalan:

Dear all,
I am a PhD student doing structural studies on a few proteins from 
Mycobacterium tuberculosis. The gene encoding the proteins I work on are cloned 
into pet22b with c terminal His tag. the proteins are expressing well. upon 
purification I am getting good yield of protein but during dialysis, the 
proteins precipitate. Kindly suggest some solutions to avoid aggregation. pI of 
one protein is 9.7 and that of the other is 5.6
I am using 25mM Tris pH 7.5 and 100 mM NaCl buffer with 5mM 
beta-mercaptoethanol and 0.5% triton x 100 for lysis, the same buffer with 
20-30mM imidazole for washing and 300mM imidazole for eluting the proteins.

Thank you
Regards
Akila

--
Akilandeswari G




--
Dr.math. et dis. nat. Jeroen R. Mesters
Deputy, Senior Researcher & Lecturer
Program Coordinator /Infection Biology/ 


Institute of Biochemistry, University of Lübeck
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phone: +49-451-31013105 (secretariate -31013101)
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http://www.uni-luebeck.de/studium/studiengaenge/infection-biology
http://www.iobcr.org 

Visiting Professorship in Biophysics, University of South Bohemia (CZ)
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It is invariably the case that high resolution X-ray structures show significantly 
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shifts, and proton chemical shifts, than the corresponding NMR structures, including 
the very best ones. Hence, in most cases, a high-resolution crystal structure (< 
2.0 Å)will provide a better description of the structure in solution than the 
corresponding NMR structure  (Kuszewski, Gronenborn & Clore, 1996, Protein Science 
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Re: [ccp4bb] software or tool for Individual residue in a Ramachandran plot graph?

[Edward A. Berry]

Thanks!

On 03/25/2017 03:36 PM, Paul Emsley wrote:

On 24/03/2017 03:39, Alex Lee wrote:

Dear All,

Is there a tool or software which can give Ramachandran information of 
individual residues
in a plot?

I used Coot to check for Ramachandran plots, but it shows all the residues in a 
coordinate I
put in Coot, not individual one. I also use "residue info" in coot, it tells 
Ramachandran
"phi psi" angles of individual residue, but it does not show it in a plot, only 
numbers.


Put this in your ~/.coot-preferences directory and when you want to Rama-check 
a residue,
bring it to the centre of the screen, click on Rama -> Rama Plot for this 
Residue.

Paul.

Re: [ccp4bb] software or tool for Individual residue in a Ramachandran plot graph?

[Edward A. Berry]

On 03/23/2017 11:39 PM, Alex Lee wrote:

Dear All,

Is there a tool or software which can give Ramachandran information of 
individual residues in a plot?

I used Coot to check for Ramachandran plots, but it shows all the residues in a coordinate I put in 
Coot, not individual one. I also use "residue info" in coot, it tells Ramachandran 
"phi psi" angles of individual residue, but it does not show it in a plot, only numbers.

Thanks ahead for any input.


Well, you can always AWK out 3 residues and run procheck on them,
something like:

  awk '$1~/ATOM/ && $5~/B/ && $6~/^6[567]/' /a/pdb/pdb2h88.ent >w.pdb
  procheck w.pdb 1.8
followed by:
  gs w_01.ps
or:
  ps2pdf w_01.ps
  acroread !$

(The AWK command will have to be tweaked if fields run together in the
  pdb due to large numbers or alt. conformations for these three residues)

I agree such a feature would be useful in coot. Sometimes you want to
know if an outlier is just outside or way outside, or if it is halfway
between two allowed regions, and with large, low-quality structures it is
 hard to find the residue in the crowded rama plot.
Clickable points in the R plot fills the bill going one way, now highlighting
points corresponding to selected residue would be the other half.
eab


w_01.pdf
Description: Adobe PDF document

Re: [ccp4bb] making a paper model

[Edward A. Berry]

On 03/09/2017 11:45 AM, Alice Dawson (Staff) wrote:

you can find a paper model of GFP at the RCSB PDB
https://cdn.rcsb.org/pdb101/learn/resources/gfp-model.pdf



Hmm- that seems to involve quite a bit of cut-and-pasting too - or rather 
cut-and taping!

2D topology diagrams are really schematics, to show the connections of the
secondary structure elements, the arrangement of strands within sheets,
and perhaps of helices within a helix bundle.
For a more complex protein than GFP, putting these secondary structure
assemblies together in a way that somehow reflects their position in the
3D structure is a very objective process, not readily amenable to
computational automation without a lot of artificial intelligence.



On 09/03/2017, 16:18, "CCP4 bulletin board on behalf of Alice Clark"
 wrote:


Dear All,
How can I get a 2D net diagram from a 3D PDB structure, to make a paper
model?

What I want to do is: take a 3D structure (GFP for Eg, PDB:4xow) make
a 2D image, print it on paper, roll it up to give the 3D structure of
the beta barrel - made of paper.

I have tried conventional topology programs but they "straighten" the
strands, so when you roll it up, the strands do not curve round the
barrel as they should. I can force this manually (cut and paste etc) -
but was hoping someone knows a way of doing it, in one step, within a
program. Perhaps using a 2D net image generator or similar?

All the best,
Alice



The University of Dundee is a registered Scottish Charity, No: SC015096

Re: [ccp4bb] CM Sephadex C50

[Edward A. Berry]

Yes, CM-Sephadex expands and contracts incredibly. For other than batch methods 
you will be much better using CM-Sepharose. I guess it is cross-linked 
sepharose, and doesn't swell up much even in distilled water.

What is happening is the negative charges of the carboxy groups repel each other, causing 
the swelling. In high salt the charges are "screened" or neutralized by 
counterions (e.g. Na+).
If you want to test your column, try with a colored protein. Cytochrome c 
sticks very tightly in 50 mM or less KPi 7.5, moves slower than the buffer 
between 50 and ~200 mM KPi, faster with increasing ionic strength. So you see a 
very tight band of bright red during loading, which diffuses and starts to move 
down during elution.



On 02/08/2017 01:47 AM, syed ibrahim wrote:

Hello All

I am using CM Sephadex C50 column for my protein. I equilibrated column with 
MOPS buffer pH 6.0. But during elution I am using only NaCl2. This looks like 
changing the bed height. Before starting elution the bed height was around 
60cm. After starting elution the bed height decreased and came upto 12 cm only. 
After 1M NaCl elution I cleaned the column with ddH2O. The column begin to 
enlarge to the height of more than 60cm. I dont understand this behaviour.

More over I could not find my protein as well. Similar situation arised even if 
I change the buffer (Acetate buffer).
Any suggestions?

Thank you

Syed

Re: [ccp4bb] advances in multisubunit membrane protein

[Edward A. Berry]

Speaking specifically for succinate dehydrogenase, there are a number of
assembly factors required for insertion of flavin, iron-sulfur clusters
(SDHAF1,2,3 . . .).  Since E. coli or Pischia make their own SDH, there is
a possibility the endogenous assembly factors and co-factors would work
with the heterogenous proteins. Not a sure thing though, so don't ask NIH
to fund it until after you've got it working!
eab


On 02/03/2017 03:46 AM, Fulvio Saccoccia, Sapienza wrote:

Dear ccp4ers,

I was wondering about most recent advances in production and
crystallization of complexes of multisubunit membrane proteins, for
instance succinate dehydrogenase, pyruvate dehydrogenase complex or even
photosystem complex. As far as I know, many large complexes of membrane
proteins were produced by tittues extraction rather than by recombinant
expression but I want to know if there is room to work with these large
assemblies by using recombinant proteins.

Best wishes


Fulvio

Re: [ccp4bb] intermolecular dissulphides

[Edward A. Berry]

Does this count as an example?
grep SSBOND /a/pdb/pdb4e9m.ent
SSBOND   1 CYS A   39CYS B   39  1555   1555  2.05
SSBOND   2 CYS C   39CYS D   39  1555   1555  2.04
SSBOND   3 CYS E   39CYS F   39  1555   1555  2.03

The A.U. contains three domain-swapped dimers. The cys are not on the swapped 
helix
but the swapping fortuitously brings the same cys in the two molecules into 
proximity
to make a disulfide. There are two or three other x-ray structures that show 
the same
domain-swapped, disulfide-clinched dimer in different packing. However an NMR 
structure
shows it to be monomeric in solution, based on estimated tumbling speed since
nmr restraints might not distinguish inter- from intramolecular contacts in a
domain-swapped dimer.
The cys is not conserved, and although this protein is expected to oligomerize,
the putative oligomerization domain is not included in this construct.

On 02/01/2017 10:17 AM, Eleanor Dodson wrote:

Does anyone know of examples of these?
I have found one - 2WQW with these SSBOND records
2WQW
SSBOND   1 CYS A  206CYS A  227  1555   6556  2.07
SSBOND   2 CYS B  206CYS B  227  1555   5556  2.15

We seem to have one but it would have to form after crystalisation?

Eleanor

Re: [ccp4bb] !SPAM: [ccp4bb] secondary structure assignment to PDB file

[Edward A. Berry]

As I understood the problem, it is that automatic assignment comes out 
differently for different structures of the same protein or proteins so close 
that they should have the same secondary structure, due to differences in 
quality of the structures. The question then is not how to determine secondary 
structure more accurately, but how to override automatic secondary structure in 
the graphics program and assign secondary structure from the best structure to 
all the others.

And the answer may be in the wording of the question: "assign secondary structure to 
the PDB file" (emphasis on FILE). If the PDB file has HELIX and SHEET records I 
believe most applications will respect them rather than re-determining secondary 
structure. So if you have a high resolution structure file that has helix and sheet 
records, copy them to the other structures. If their is only one structure that is 
getting assigned differently in different applications, check if it has HELIX and SHEET 
records, and if not edit them in according to your best idea of the secondary structure.


On 01/30/2017 04:08 AM, Tim Gruene wrote:

Dear J. Vitali,

there are tools that have been mentioned by others, that assist with the
assignment. The final decision depends on you as researcher. You should
visually check the hydrogen bonding whether the boundaries are consistent with
the automated assignment.

People often seem to think that secondary structure is program driven, but
it's your structure that provides that data, and the researcher to make the
final decision.

Kind regards,
Tim

On Sunday 29 January 2017 11:41:49 AM chemocev marker wrote:

Hi
Is there any tool that can assign secondary structure to the PDB file. The
problem is if I used different modelling tools, there are regions in the
protein which does not remain consistent and looks different in different
application.

best

J. Vitali

Re: [ccp4bb] Unknown electron density blob, pdb convention for partially ordered ligands

[Edward A. Berry]

Uma's use of quotes around "di" suggests a related question about PDB 
convention. It was my (perhaps not very good) understanding that ligands should be 
identified by what is actually present in the crystal, and not by what can be modeled. 
For example endogenous ubiquinone is likely to be UQ50 (depending on the species) but 
most of that 50-carbon side chain is hanging out in the lipid or detergent and completely 
disordered. Still we should use the ligand identifier for UQ50, even though codes exist 
for UQ with 5 or 10-carbon side chains that are much better accommodated by the density.

If that is the case, one should not use the pdb identifier for diethylene glycol (PEG) 
when PEG4k was the precipitant, unless you believe that the binding site has specifically 
selected diethylene glycol from an extremely broad range of polymer lengths in the added 
material.  Using the identifier for a much longer PEG will result in a large number of 
"missing atoms" listed in the report, but would eliminate the unreasonable 
assumption that PEG fragment models must always end with a terminal oxygen.

Even if that is the rule, I would agree that PEGs would be a good place to 
ignore the rule. Since PEGs have a MW distribution, it is impossible to know 
exactly what is bound and it may be different in different unit cells. If you 
are not going to get it right no matter what you put, you might as well put 
something that fits.
eab

On 01/25/2017 09:51 AM, Uma Gabale wrote:

Dear all,
Thank you very much for your replies. It is a PEG, a "di"ethylene glycol to be 
precise, in most chains.
Best regards,
Uma.
--
Uma Gabale, PhD
Research Associate
Molecular and Cellular Biochemistry
Indiana University Bloomington

Re: [ccp4bb] help with Buccaneer

[Edward A. Berry]

When faced with a program requiring HL coefficients, and having
phases from a (partial) model, I used to run a ccp4 program called
sigmaa to generate the HL coefficients. I'm not sure if this is
theoretically a good idea, but at least it allows the program to run.

On 01/19/2017 11:10 PM, Vikram Dalal wrote:

Hi Everyone,

I want to run the Buccaneer. I need Hendrickson-Lattman coefficients for it. 
How I can find Hendrickson-Lattman coefficients?


*
*

Thanks & Regards,

Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent Count Numbers

[Edward A. Berry]

On 11/30/2016 10:16 PM, Keller, Jacob wrote:

If you fine slice and everything is then a partial, isn't that *more* sensitive 
to lack of synchronization between the shutter and rotation axis than the 
wide-frame method where there's a larger proportion of fulls that don't 
approach the frame edges (in rotation space) ?  Especially if you're 3D profile 
fitting ?


That is how the argument seems to go in Pflugrath 1999, but I would think that 
shutter jitter is a random error, so it would seem better to have several of 
these random errors for a given spot than just one. Perhaps measuring with high 
multiplicity would have the same averaging effect.


Is fine slicing more or less beneficial at high resolutions relative to lower 
ones ?


In terms of I/sigI, it seems to be the same proportional improvement across all 
resolutions. See Fig 4 of the Pflugrath 1999 paper.

JPK


I think the problem there is that, if the shutter jitter is random with a 
constant sigma, it becomes a larger percent of the total exposure for that 
frame. It would be like taking a 1ml pipetor with an error of 2% of full scale, 
i.e. 20 ul. Because you want to average this out, you set it to 200 ul and 
pipet 5 times. The sigma of that measurement would be sqrt(5) * 20 ul, I think, 
so worse than doing it all in one shot. On the other hand if you take a 200 ul 
pipet with sigma 2% of full scale or 4 ul, and take 5 times, the error is 
sqrt(5) * 4 ul which is less than 20 ul.
Of course this would not apply to reflections that are fully recorded on one 
frame since they are not reflecting while the shutter is open/closing. Then it 
would be only variation in background.



Phil Jeffrey

Princeton

--

*From:*CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
*Sent:* Wednesday, November 30, 2016 5:44 PM
*To:* CCP4BB@JISCMAIL.AC.UK 
*Subject:* Re: [ccp4bb] Effects of Multiplicity and Fine Phi with Equivalent 
Count Numbers

If the mosaicity is, say, 0.5 deg, and one is measuring 1 deg frames, about 
half the time is spent measuring non-spot background noise under spots in phi, 
which is all lumped into the intensity measurement. Fine slicing reduces this. 
But I am conjecturing that there is also fine-slicing-mediated improvement due 
to averaging out things like shutter jitter, which would also be averaged out 
through plain ol’ multiplicity.

I guess a third equal-count dataset would be useful as well: one sweep with 
six-fold finer slicing. So it would be:

One sweep, 0.6 deg, 60s

Six sweeps, 0.6 deg, 10s

One sweep, 0.1 deg, 10s

Or something roughly similar. Who will arrange the bets?

JPK

*From:*Boaz Shaanan [mailto:bshaa...@bgu.ac.il]
*Sent:* Wednesday, November 30, 2016 5:19 PM
*To:* Keller, Jacob >; 
CCP4BB@JISCMAIL.AC.UK 
*Subject:* RE: Effects of Multiplicity and Fine Phi with Equivalent Count 
Numbers

Hi Jacob,

I may have missed completely your point but as far as my memory goes, the main 
argument in favour of fine slicing has always been reduction of the noise 
arising from incoherent scattering, which in the old days arose from the 
capillary, solvent, air, you name it. The noise reduction in fine slicing is 
achieved by shortening the exposure time per frame. This argument still holds 
today although the sources of incoherent scattering could be different. Of 
course, there are other reasons to go for fine slicing such as long axes and 
others. In any case it's the recommended method these days, and for good 
reasons, isn't it?

   Best regards,

Boaz

/Boaz Shaanan, Ph.D. //
/Dept. of Life Sciences /
/Ben-Gurion University of the Negev /
/Beer-Sheva 84105 /
/Israel /
//
/E-mail: bshaa...@bgu.ac.il /
/Phone: 972-8-647-2220  Skype: boaz.shaanan /
/Fax:   972-8-647-2992 or 972-8-646-1710 //

//

Re: [ccp4bb] AW: AW: AW: [ccp4bb] confusing crystal diffraction

[Edward A. Berry]

On 11/17/2016 08:36 AM, herman.schreu...@sanofi.com wrote:

Dear Shijun,

The reject.hkl file is the file with all rejected reflections. The first three 
numbers are h, k and l. For the other items you have to consult the HKL manual. 
As I said, I am not familiar with HKL2000. However, in your case, I would look 
in the log files instead of the .hkl files and see if you can find somewhere 
how many reflections were rejected for what reason. With XDS, I know where to 
find these numbers, for HKL2000 you have to ask a HKL2000 expert. For that 
reason I CC’d this email to the bulletin board.

Best,

Herman

The log file has a table listing rejected reflections for each frame.
I need to consult the manual for the meaning of #2 "zero sigma or profile test" 
-
 Others are pretty self-explanatory.

 1 - count of observations deleted manually
 2 - count of observations deleted due to zero sigma or profile test
 3 - count of non-complete profiles (e.g. overloaded) observations
 4 - count of observations deleted due to sigma cutoff
 5 - count of observations deleted below low resolution limit,
 6 - count of observations deleted above high resolution limit,
 7 - count of partial observations
 8 - count of fully recorded observations used in scaling

  12345678
 IP fitted, no o 1  0.92700.320 4481  218 20233 1173 7665 2294
 IP fitted, no o 2  1.03860.280 2885  147 13313  913 3975 2349
 IP fitted, no o 3  0.96850.200 3014  165 13614  962 4698 2353
 IP fitted, no o 4  1.0.000 2912  124 13455 1061 4250 2355
 IP fitted, no o 5  0.9894   -0.250 2924  139 13332 1055 4359 2301
 IP fitted, no o 6  0.9720   -0.540 2639  136 12143 1133 4242 2370
 IP fitted, no o 7  0.9489   -0.890 2848  127 12753 1118 4509 2284
 IP fitted, no o 8  0.9700   -1.300 3018  145 13161 1194 4722 2380
 IP fitted, no o 9  0.9275   -1.680 2568  136 11750 1227 4111 2331
 IP fitted, no o10  0.9619   -2.180 2597  144 11730 1261 4617 2293
 IP fitted, no o11  0.9295   -2.710 2491  135 10890 1233 4194 2335
 IP fitted, no o12  0.9589   -3.250 2713  155 11940 1265 4661 2375
 IP fitted, no o13  0.9533   -3.770 2548  167 11102 1347 4544 2263
 IP fitted, no o14  0.9683   -4.370 2413  170 10673 1393 4475 2322
 IP fitted, no o15  0.9639   -4.920 2420  141 10904 1340 4162 2357
 IP fitted, no o16  0.9386   -5.440 2480  167 10243 1337 4357 2403
 IP fitted, no o17  0.9576   -5.970 2400  144 10433 1373 4317 2378




*Von:*张士军[mailto:21620150150...@stu.xmu.edu.cn]
*Gesendet:* Donnerstag, 17. November 2016 13:41
*An:* Schreuder, Herman R/DE
*Betreff:* Re: AW: AW: [ccp4bb] confusing crystal diffraction


Dear Herman

This is the rejection file ,I can not understand that.Does anyone familiar with 
HKL2000 tell me what those mean ?
reject hkl
   10   13 p+   254   4.7   68.4  101  479.1
   10   13 a+   255   4.7 3117.1  101  479.1
   10   13 p+   256   4.7  263.7  101  479.1
   0   -1   13 n+   268   6.2   42.3  101  495.4
   0   -1   13 a+   269   6.2 3330.3  101  495.4
   0   -1   13 p+   270   6.2  881.7  101  495.4
  -10  -13 n-   251   4.5   20.2  101  491.1
  -10  -13 a-   252   4.5 2940.5  101  491.1
  -10  -13 p-   253   4.5  460.7  101  491.1
   01  -13 a-89   6.6 2858.2  101  343.0
   01  -13 p-90   6.6  612.6  101  343.0
   01  -13 p-   264   6.1   66.3  101  525.5
   01  -13 a-   265   6.1 3533.4  101  525.5
   01  -13 p-   266   6.1  796.9  101  525.5
   0   -1   15 p+81   4.6  786.8  101  247.5
   0   -1   15 a+82   4.6  916.5  101  247.5
   01  -15 n-85   5.1   15.5  101  229.2
   01  -15 a-86   5.1 1516.5  101  229.2
   01  -15 p-87   5.1  194.3  101  229.2
   11   -8 n-   275   5.30.5  101  136.4
   11   -8 p-   276   5.3   34.2  101  136.4
   11   -8 p-   277   5.3  260.0  101  136.4
   11   -8 a-   278   5.3  517.6  101  136.4
   11   -8 p-   279   5.3  473.3  101  136.4
   11   -8 p-   280   5.3  203.4  101  136.4
   11   -8 p-   281   5.3   78.0  101  136.4
   2   -17 n+   285   4.4   32.2  101  280.7
   2   -17 a+   286   4.4 1731.7  101  280.7
   2   -17 p+   287   4.4  250.6  101  280.7

-原始邮件-
*发件人:* herman.schreu...@sanofi.com 

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

[Edward A. Berry]

What about China? Singapore?

On 11/09/2016 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] determine NCS operators from PDB coordinates

[Edward A. Berry]

Well done.
Sorry for the many typos, but they don't seem to have slowed you down any!
eab

On 07/07/2015 11:25 AM, Tobias Beck wrote:

Dear all,

Thanks for your helpful emails.

Here is a short summary:

I followed Edward Berry's suggestions. Unfortunately I could not use any 
superposition to the new structure, as suggested by some, since the structures 
are too different.
I used lsqman and mama to obtain the sperical polars for my twofold as pointed 
out by Ed. After this, I rotated my structure in two steps based on these 
angles, but using rotation matrixes in the form

cos_theta   -sin_theta0

sin_thetacos_theta0

0   0  1

as pointed out by Ed, to align the twofold axis in my structure to a twofold 
axis in the new space group. That was done in pdbset (I did not need to 
consider translation in my case).

Thanks again for helping me!

Best wishes, Tobias.

On Fri, Jun 19, 2015 at 4:54 PM, Edward A. Berry ber...@upstate.edu 
mailto:ber...@upstate.edu wrote:

A number of superposition programs allow to superimpose specified atoms 
(such as CA).
Once you get the operator, comparing two different operators is not a job
for a conventional superposition program, since you are superimposing a
line on a line which has the extra degree of freedom- rotation about the 
line.
If you express the operator in spherical polar coordinates, which the 
superposition
program may provide or you can get from the matrix using ccp4 rotmat,
you should be able to work out the relation between the axes.

Using the Uppsala sofware factory programs:

#This is superimposing parts of chains C, A, B on P, N, O (and vice versa - 
it's proper 2-fold)
lsqman -b eof
chain_mode original
re m1  cbc596.pdb
exp m1
C20-370  P20-370 A20-200 A250-400 B30-200 B250-400 N20-200 N250-400 O30-200 
O250-400
m1
P20  C20 N20 N250 O30 O250 A20 A250 B30 B250
save m1 m1 ncsasc01.odb
quit
eof

This prints the operator and saves it in ncsasc01.odb*

- The   2010 atoms have an RMS distance of0.130 A
- RMS delta B  =   15.367 A2
- Corr. coeff. =  0.6601
- Rotation:  -0.834671 tel:0.834671 -0.550747 -0.001321 
tel:0.550747%20-0.001321
--0.550747 tel:0.550747 0.834660 0.004400 
tel:0.834660%20%200.004400
--0.001321  0.004400 -0.89 tel:0.89
- Translation :129.38438.428   171.594
---

Now use mama in an off-label way to convert to polar coordinates:
mama
overlap ncs  ncsasc01.odb
gives:
---
- RT-OP  1 =-0.8346710 tel:0.8346710   -0.5507473 tel:0.5507473   
-0.0013208129.384
-   -0.5507473 tel:0.5507473 0.8346604 tel:0.8346604
0.0044000 38.428
-   -0.00132080.0044000   -0.894 tel:0.894
171.594
- Determinant of rotation matrix 1.00
- Column-vector products (12,13,23)  0.000.000.00
- Crowther Alpha Beta Gamma   106.709 179.736  73.291
- ***Spherical polars Omega Phi Chi   90.132 -73.291 
180.000***
- Direction cosines of rotation axis 0.287514 tel:0.287514   -0.957774 
-0.002303 tel:0.957774%20%20%20-0.002303
- X-PLOR polars Phi Psi Kappa *undefined* *undefined* 180.000
- Lattmann Theta+ Theta2 Theta-  -180.000 179.736-146.582
- Rotation angle 180.000
-
  * if you use the .odb file outside of the USF software, be aware the 
matrix is the transpose
(or more accurately it is written by columns). In ccp4 this is taken care of withthe 
keyword odb.


On 06/19/2015 09:07 AM, Tobias Beck wrote:

Dear all,

I have a PDB file that contains NCS in the asymmetric unit, probably 
point group D3.

1.) What program is recommended for determining the symmetry operators 
from PDB coordinates? I found findncs, but this uses only heavy atom 
coordinates (I could probably use just the sulfurs from the PDB as a work 
around).

2.) Then I would like to compare the PDB file to a related structure. 
Here I would like to align the symmetry operators determined above with 
symmetry elements found in a different space group, for example align the 
twofold axis from NCS with a twofold axis in found in a particular space group.
What is a good way to go about this?

I am aware that NCS is used in programs as restraints during 
refinement, but here I am interested in obtaining the NCS symmetry operators 
and aligning them to symmetry elements present in a new space group. Maybe I am 
overlooking an obvious solution.

Any help is greatly appreciated.

Thanks and best wishes, Tobias

Re: [ccp4bb] topdraw

[Edward A. Berry]

I think it is part of ccp4:
$CCP4/bin/topdraw

On 07/05/2015 10:30 AM, Faisal Tarique wrote:

Dear all

Can anybody provide me the link to download or install  TopDraw  a topology 
drawing interface in CCP4..?

Thanks

--
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] paired refinement

[Edward A. Berry]

Another criterion for cutoff, also requiring the structure to be solved,
is the agreement between data and structure, e.g. Rfree or CCfree.
I think it is very unlikely that you could get Rfree =.2493 in a shell
which contains only noise. So I would suggest doing paired refinement
to 2.2 and 2.1 A (if the data is available).

On 07/01/2015 07:15 PM, Eric Karg wrote:

Hi all,

I have a dataset processed in XDS to 2.3 A (based on CC1/2). I'm trying to do 
paired refinement to determine the optimal resolution cutoff. Here is what I 
get at different resolutions set in Phenix:

Final Rfree/Rwork:
2.7— 0.2498/0.2027
2.6— 0.2519/0.2009
2.5— 0.2567/0.2025
2.4 — 0.2481/0.2042
2.3 — 0.2493/0.2075

The geometry of all output structures are similar.

1. What is the high resolution cutoff based on these data? I know that Rfree/Rwork 
at different resolution should not be compared, but is there a simple way to do the 
test as described in the KD 2012 Science paper using Phenix GUI?

2. For refining a structure at a lower resolution (lower than the initial 
dataset), do I simply set the resolution limit in the refinement or I need to 
reprocess the data starting from the images? Do I need to do anything with 
Rfree flags? Based on the discussions on this forum I know I should deposit the 
highest resolution dataset but my question is about the mtz file which will be 
used for refinement.

Thank you very much for your help!

Re: [ccp4bb] paired refinement

[Edward A. Berry]

My take on this-
No one has been willing to specify a cutoff (and probably there is no rigorous 
way to
mathematically define the cutoff) and say If CC* (or CCfree or whatever) is 
below X
then it will not improve your structure, if above X then it will. Probably 
depends
among other things on how strong the lower resolution data is, how good the
structure is without the added data.
On the other hand in paired refinement, if adding the data improves the 
structure
as measured by Rfree in a zone excluding the added data, then it is hard to deny
that that data are worth including.

eab

On 07/02/2015 12:52 PM, Keller, Jacob wrote:

Wasn’t all of this put to bed through the implementation of CC measures?

JPK

*From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Robbie 
Joosten
*Sent:* Thursday, July 02, 2015 12:46 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* Re: [ccp4bb] paired refinement

But it is not the R-free of the shell here. In paired refinement you take the 
R-free of the reflections outside the shell.

Cheers,
Robbie

Sent with my Windows Phone

--

*Van: *Edward A. Berry mailto:ber...@upstate.edu
*Verzonden: *‎2-‎7-‎2015 18:43
*Aan: *CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK
*Onderwerp: *Re: [ccp4bb] paired refinement

Another criterion for cutoff, also requiring the structure to be solved,
is the agreement between data and structure, e.g. Rfree or CCfree.
I think it is very unlikely that you could get Rfree =.2493 in a shell
which contains only noise. So I would suggest doing paired refinement
to 2.2 and 2.1 A (if the data is available).

On 07/01/2015 07:15 PM, Eric Karg wrote:
  Hi all,
 
  I have a dataset processed in XDS to 2.3 A (based on CC1/2). I'm trying to do 
paired refinement to determine the optimal resolution cutoff. Here is what I get 
at different resolutions set in Phenix:
 
  Final Rfree/Rwork:
  2.7— 0.2498/0.2027
  2.6— 0.2519/0.2009
  2.5— 0.2567/0.2025
  2.4 — 0.2481/0.2042
  2.3 — 0.2493/0.2075
 
  The geometry of all output structures are similar.
 
  1. What is the high resolution cutoff based on these data? I know that 
Rfree/Rwork at different resolution should not be compared, but is there a simple way 
to do the test as described in the KD 2012 Science paper using Phenix GUI?
 
  2. For refining a structure at a lower resolution (lower than the initial 
dataset), do I simply set the resolution limit in the refinement or I need to 
reprocess the data starting from the images? Do I need to do anything with Rfree 
flags? Based on the discussions on this forum I know I should deposit the highest 
resolution dataset but my question is about the mtz file which will be used for 
refinement.
 
  Thank you very much for your help!
 

Re: [ccp4bb] paired refinement

[Edward A. Berry]

Yes, my stupid mistake.  Please delete/disregard!

On 07/02/2015 12:46 PM, Robbie Joosten wrote:

But it is not the R-free of the shell here. In paired refinement you take the 
R-free of the reflections outside the shell.

Cheers,
Robbie

Sent with my Windows Phone
--
Van: Edward A. Berry mailto:ber...@upstate.edu
Verzonden: ‎2-‎7-‎2015 18:43
Aan: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK
Onderwerp: Re: [ccp4bb] paired refinement

Another criterion for cutoff, also requiring the structure to be solved,
is the agreement between data and structure, e.g. Rfree or CCfree.
I think it is very unlikely that you could get Rfree =.2493 in a shell
which contains only noise. So I would suggest doing paired refinement
to 2.2 and 2.1 A (if the data is available).

On 07/01/2015 07:15 PM, Eric Karg wrote:
  Hi all,
 
  I have a dataset processed in XDS to 2.3 A (based on CC1/2). I'm trying to do 
paired refinement to determine the optimal resolution cutoff. Here is what I get 
at different resolutions set in Phenix:
 
  Final Rfree/Rwork:
  2.7— 0.2498/0.2027
  2.6— 0.2519/0.2009
  2.5— 0.2567/0.2025
  2.4 — 0.2481/0.2042
  2.3 — 0.2493/0.2075
 
  The geometry of all output structures are similar.
 
  1. What is the high resolution cutoff based on these data? I know that 
Rfree/Rwork at different resolution should not be compared, but is there a simple way 
to do the test as described in the KD 2012 Science paper using Phenix GUI?
 
  2. For refining a structure at a lower resolution (lower than the initial 
dataset), do I simply set the resolution limit in the refinement or I need to 
reprocess the data starting from the images? Do I need to do anything with Rfree 
flags? Based on the discussions on this forum I know I should deposit the highest 
resolution dataset but my question is about the mtz file which will be used for 
refinement.
 
  Thank you very much for your help!
 

Re: [ccp4bb] [Fwd: Re: [ccp4bb] FW: New ligand 3-letter code (help-7071)]

[Edward A. Berry]

  I can't imagine a journal doing that can you?  When I work on my
supplementary material in a paper I don't expect that the journal will
take a bit out and publish it separately to support the work of my
competitors. Not out of spite that I was beaten - but because I don't
want to take the responsibility for checking their science for them!


I don't see the problem here. What about the dozens of authors who
will benefit from using your ligand in their structure _after_ your
structure comes out? You don't take responsibility for checking their
science. Every author gets a copy of his final structure to check
before it is released and each is responsible for his own.
The only difference here is whether the competitor got to use it first,
(which might sting a bit) or only after you had already made it your
own with the first structure.

I guess the ligand database is the responsibility of the pdb, but
they depend on first depositors to help set up each ligand, so
it is not surprising if the type model has coordinates from the
first depositor's structure (although it would be convenient if
they were all moved to c.o.m. at 0,0,0). When another group publishes
a structure with the ligand, they will not be publishing the first
depositor's coordinates because the ligand will be moved to its position
in their structure and refined against their data, probably with
somewhat different restraints.

If the ligand is a top secret novel drug lead that your company is
developing I guess it would come as a shock to find someone else has
already deposited it, and it might be good to hasten not the
publication but protecting of the compound with a patent!

Although Miriam says a new 3-letter code is generated when no match is found,
I believe the depositor's code will be used if it is available,
at least one of mine was last year, so there is some use for Nigel's
utility if you want to stamp your new compound with a rememberable name.

eab

On 06/21/2015 06:33 PM, Martyn Symmons wrote:

Miri raises important points about issues in the PDB Chemical
Component Dictionary - I think part of the problem is that this is
published completely separately from the actual PDB - so for example I
don't think we have an archive of the CCD for comparison alongside the
PDB snapshots? This makes it difficult to follow the convoluted track
of particular ligands through the PDB's many,many changes to small
molecule definitions.

But following discussion with other contributors offline I want to
make it clear what is my understanding of the ZA3 (2Y2I /2Y59) case:

I am clear there was no unethical behaviour by either group in the
course of their work on these structures and the publication of them.

The problem I am highlighting is that the PDB don't understand
publishing ethics - what happened in ZA3 was that they published a
little bit of one group's work to support the work of someone who was
scooping them!

  I can't imagine a journal doing that can you?  When I work on my
supplementary material in a paper I don't expect that the journal will
take a bit out and publish it separately to support the work of my
competitors. Not out of spite that I was beaten - but because I don't
want to take the responsibility for checking their science for them!

All the best
   Martyn

Cambridge

On Sun, Jun 21, 2015 at 7:01 PM, Miri Hirshberg
02897e8e9f0f-dmarc-requ...@jiscmail.ac.uk wrote:

Sun., June 21st 2015

Good evening,

adding several general points to the thread.

(1) Fundamentally PDB unlike other chemical databases
insists that all equal structures should have the same 3-letter
code and the same atom names - obviously for amino acids and say ATP.

  (1.1) Needless to say there are endless examples in the PDB of two
ligands differ by let say one hydroxyl group, where equivalent atoms in
the two ligands having totally different names.

(2) When a structure is deposited with a ligand, the ligand is first
compared against PDB chem_comp database (CCD) and against the on-hold
chem_comp (CCD) (naturally the latter is not publicly available),
and only if no-match can be found a new three-letter code  is generated
and assigned.

If not, then this is a mistake in annotation and should not happen.

(3) Exception to the above take several different flavours. This
include:

  (3.1) When the same ligand is described in PDB as a 3-letters-code
and as well as a combination of two different 3-letters-code ligands.
An example out of many is phosphoserine. The 3-letter-code
in PDB CCD is SEP which is used in 704 PDB entries (RCSB counting
21-June-2015). But in the PDB entry 3uw2 the phosphoserine 109A is
described as a combination of SER and the inorganic phosphate PO4 !!!
(a side point: note the inorganic PO4 became organic upon this linkage -
a PDB chemical conundrum!!).

  (3.2) CCDC does not make any attempt to standardise atom names nor to
match same structures to have equal atom names - original author atom
names are kept so that amino acids may have 

Re: [ccp4bb] FW: New ligand 3-letter code (help-7071)

[Edward A. Berry]

You raise some good points, but as far as better confidentiality 
pre-publication goes. unreleased entries are not secret in any case- if the 
second group is at all nervous about competition, they will be searching the 
unreleased entries database (http://www.rcsb.org/pdb/search/searchStatus.do), 
with the name of their protein or ligand, or suspected competing authors names, 
on a weekly basis.
We deposited 1yq3 and 1yq4 in Feb 2005, well before we were ready to 
publish. Regularly searching our protein in the unreleased entries, we saw 1zoy 
and 1zp0 deposited in May. And even so,they beat us to publication, but with 
lower resolution structures. I sometimes wonder if they would have waited for 
better data had they not known we were about to publish.

Elsewhere in the thread: I don't think you want to call your new ligand UNL. 
From what I understand that indicates the authors were unable to identify it, 
and by default it will remain UNL in the final released entry (although some 
back-and forth with the annotator would put things right).

eab

On 06/19/2015 08:38 PM, Martyn Symmons wrote:

By oversimplifying the situation here the PDB does not answer my
related point about competing crystallographers:
My scenario:

Group A deposits structure with new drug - gets their three-letter
code for example ZA3
  they then get to check the coordinates and chemical definition of this ligand.

But suppose a little after that a competing group B deposits their
structure with the same drug which they think is novel - but no...
they get assigned the now described ZA3 which has been checked by the
other group.

  Then it is a race to see who gets to publish and release first. And
if it is the second group B who wins then they are publishing the work
of their A competitors - who have done the depositing and checking of
the ligand  description.

  Sounds unlikely? Well, it actually happened in 2011 for my exact
example ZA3 - present in 2Y2I and in 2Y59 from competing groups.

  From the dates in the mmcif it was 2Y2I depositors who set up and had
a chance to review the description of ZA3 ligand. Only to see it
released a week before their crystal structure, when their ZA3
appeared to accompany competing 2Y59! It is amazing that the PDB did
not spot this and arrange a suitable workaround.

Just to check:
mmcif for ZA3 shows it was created for 2Y2I:
...
_chem_comp.pdbx_model_coordinates_db_code2Y2I
...
But it was modified for release:
...
_chem_comp.pdbx_modified_date2011-07-22
...
corresponding to the early 2011-07-27 release date of the competing
structure: 2Y59 even though this PDB was  _deposited_ second.

The ZA3 ligand definition released with 2Y59 actually embodies the
atomic coordinates from the 2Y2I structure:

mmcif
ZA3 O6   O6   O 0  1 N N N 8.279  7.165  40.963 0.311  -1.061 -0.920
O6   ZA3 1
ZA3 C5   C5   C 0  1 N N N 9.132  8.047  40.908 0.147  -0.205 -0.073
C5   ZA3 2  ...
PDB 2Y2I
HETATM 3598  O6  ZA3 A1000   8.279   7.165  40.963  1.00 41.25   O
HETATM 3599  C5  ZA3 A1000   9.132   8.047  40.908  1.00 63.20
   C ...

Surely a better approach would be to allow both groups a chance to
work through and sign off on independent ligand descriptions?

Then whoever releases first would release both a novel structure and
the ligand definition _they_ deposited and checked. Their priority can
then be asserted and the other group contacted to ask if they agree to
accept this definition. This also has the advantage of better
confidentiality pre-publication.

Another problem from any cross-linking of definitions is that say
group A are motivated by reviewers' reports to change the definition
of ZA3 pre-release. Well now the change impinges on the chemical
meaning of other group B's deposited structure. For example ZA3 mmcif
has a statement:

ZA3 Modify aromatic_flag 2011-06-04 RCSB

so this change was pre-release - but we cannot be sure what motivated
this - whether it was signed off by the 2Y2I authors or the 2Y59
authors (or both?)

With the accelerating pace of drug discovery for sure this sort of
uncertainty is going to happen again.Unless the PDB have changed their
practice for ligand deposition?

All the best
  Martyn

Cambridge.

On Fri, Jun 19, 2015 at 1:49 PM, Sheriff, Steven steven.sher...@bms.com wrote:

All:



Since the original query was cross-posted on both the COOT mailing list and
the CCP4BB Rachel Green gave me permission to forward this to both. She
provides links about the mechanism of assignment of 3-letter codes. In the
third link below, my original suggestion to the COOT mailing list that one
could just use UNK is incorrect as that is reserved for unknown amino acids.
According to this document, I should have suggested UNL for an unknown
ligand.



Steven



From: Rachel Kramer Green [mailto:kra...@rcsb.rutgers.edu]
Sent: Tuesday, June 16, 2015 10:21 AM
To: Sheriff, Steven
Cc: info
Subject: Re: New ligand 3-letter code (help-7071)

Re: [ccp4bb] determine NCS operators from PDB coordinates

[Edward A. Berry]

A number of superposition programs allow to superimpose specified atoms (such 
as CA).
Once you get the operator, comparing two different operators is not a job
for a conventional superposition program, since you are superimposing a
line on a line which has the extra degree of freedom- rotation about the line.
If you express the operator in spherical polar coordinates, which the 
superposition
program may provide or you can get from the matrix using ccp4 rotmat,
you should be able to work out the relation between the axes.

Using the Uppsala sofware factory programs:

#This is superimposing parts of chains C, A, B on P, N, O (and vice versa - 
it's proper 2-fold)
lsqman -b eof
chain_mode original
re m1  cbc596.pdb
exp m1
C20-370  P20-370 A20-200 A250-400 B30-200 B250-400 N20-200 N250-400 O30-200 
O250-400
m1
P20  C20 N20 N250 O30 O250 A20 A250 B30 B250
save m1 m1 ncsasc01.odb
quit
eof

This prints the operator and saves it in ncsasc01.odb*

- The   2010 atoms have an RMS distance of0.130 A
- RMS delta B  =   15.367 A2
- Corr. coeff. =  0.6601
- Rotation:  -0.834671 -0.550747 -0.001321
--0.550747  0.834660  0.004400
--0.001321  0.004400 -0.89
- Translation :129.38438.428   171.594
---

Now use mama in an off-label way to convert to polar coordinates:
mama
overlap ncs  ncsasc01.odb
gives:
---
- RT-OP  1 =-0.8346710   -0.5507473   -0.0013208129.384
-   -0.55074730.83466040.0044000 38.428
-   -0.00132080.0044000   -0.894171.594
- Determinant of rotation matrix 1.00
- Column-vector products (12,13,23)  0.000.000.00
- Crowther Alpha Beta Gamma   106.709 179.736  73.291
- ***Spherical polars Omega Phi Chi   90.132 -73.291 180.000***
- Direction cosines of rotation axis 0.287514   -0.957774   -0.002303
- X-PLOR polars Phi Psi Kappa *undefined* *undefined* 180.000
- Lattmann Theta+ Theta2 Theta-  -180.000 179.736-146.582
- Rotation angle 180.000
-
 
* if you use the .odb file outside of the USF software, be aware the matrix is the transpose

(or more accurately it is written by columns). In ccp4 this is taken care of withthe 
keyword odb.

On 06/19/2015 09:07 AM, Tobias Beck wrote:

Dear all,

I have a PDB file that contains NCS in the asymmetric unit, probably point 
group D3.

1.) What program is recommended for determining the symmetry operators from PDB 
coordinates? I found findncs, but this uses only heavy atom coordinates (I 
could probably use just the sulfurs from the PDB as a work around).

2.) Then I would like to compare the PDB file to a related structure. Here I 
would like to align the symmetry operators determined above with symmetry 
elements found in a different space group, for example align the twofold axis 
from NCS with a twofold axis in found in a particular space group.
What is a good way to go about this?

I am aware that NCS is used in programs as restraints during refinement, but 
here I am interested in obtaining the NCS symmetry operators and aligning them 
to symmetry elements present in a new space group. Maybe I am overlooking an 
obvious solution.

Any help is greatly appreciated.

Thanks and best wishes, Tobias.
--
___

Dr. Tobias Beck
- independent group leader -
RWTH Aachen University
Institute of Inorganic Chemistry
Landoltweg 1, office: 304N
52056 Aachen, Germany
phone:  +49-241-80-90057
fax:   +49-241-80-99003
web: http://www.ac.rwth-aachen.de/extern/beck/
___

Re: [ccp4bb] New ligand 3-letter code

[Edward A. Berry]

Neat! It's true the PDB will choose a unique code for you,
but they will use what you supply if it is already unique,
and it is nice to be able to choose something that can help
you remember what it stands for.
Gone are the days when we could choose meaningfull pdb ID's
(like 1PRC, 2PRC, 3PRC etc are all photosynthetic reaction center),
but there is still some freedom in choosing the ligand ID's.
They are filling up fast, though!

locust 123% elbow.get_new_ligand_code AC
Unique ligand code : AC3

locust 129% elbow.get_new_ligand_code A?3
Unique ligand code : A?3

eab

On 06/06/2015 12:13 PM, Nigel Moriarty wrote:

I wrote something a while ago the finds an unused code.


% elbow.get_new_ligand_code


Unique ligand code : 7V8


% elbow.get_new_ligand_code A


Unique ligand code : A6E


Cheers

Nigel

---
Nigel W. Moriarty
Building 64R0246B, Physical Biosciences Division
Lawrence Berkeley National Laboratory
Berkeley, CA 94720-8235
Phone : 510-486-5709 Email : nwmoria...@lbl.gov
Fax   : 510-486-5909   Web  : CCI.LBL.gov http://CCI.LBL.gov

On Fri, Jun 5, 2015 at 6:59 AM, Eleanor Dodson eleanor.dod...@york.ac.uk 
mailto:eleanor.dod...@york.ac.uk wrote:


I use any 3 letter/number code that i want. If you read the corresponding 
cif file into coot it is used in preference to any in the library. The PDB 
deposition team will assign a code if it is a new ligand to the database. Could 
you relay this to original poster?

Thanks

Jim Brannigan


On 5 June 2015 at 14:58, Eleanor Dodson eleanor.dod...@york.ac.uk 
mailto:eleanor.dod...@york.ac.uk wrote:

OK - thank you.
How are things?
E


-- Forwarded message --
From: *Jim Brannigan* jim.branni...@york.ac.uk 
mailto:jim.branni...@york.ac.uk
Date: 5 June 2015 at 14:39
Subject: Re: New ligand 3-letter code
To: Eleanor Dodson eleanor.dod...@york.ac.uk 
mailto:eleanor.dod...@york.ac.uk


Hi Eleanor

I use any 3 letter/number code that i want. If you read the 
corresponding cif file into coot it is used in preference to any in the 
library. The PDB deposition team will assign a code if it is a new ligand to 
the database. Could you relay this to original poster?

Thanks

Jim Brannigan

On 5 June 2015 at 11:28, Eleanor Dodson eleanor.dod...@york.ac.uk 
mailto:eleanor.dod...@york.ac.uk wrote:

I use your method - trial  error..
It would be nice if at least there was a list somewhere of 
unassigned codes!


On 5 June 2015 at 09:16, Lau Sze Yi (SIgN) lau_sze...@immunol.a-star.edu.sg 
mailto:lau_sze...@immunol.a-star.edu.sg wrote:

Hi,

What is the proper way of generating 3-letter code for a new 
ligand? As of now, I insert my ligand in Coot using smiles string and for the 
3-letter code I picked a non-existent code by trial and error (not very 
efficient). A cif file with corresponding name which I generated using Phenix 
was imported into Coot.

I am sure there is a proper way of doing this. Appreciate your 
feedback.

Regards,
Sze Yi

Re: [ccp4bb] How many is too many free reflections?

[Edward A. Berry]

In other words, the free set for each complex must be
such that reflections that are also present in the apo dataset retain
the FreeR flag they had in that dataset.


A very easy way to achieve this- generate a complete dataset to ridiculously
high resolution with the cell of your crystal, and assign free-r flags.
(If the first structure has been already solved, merge it's free set and
extend to the new reflections)
Now for every new structure solved, discard any free set that the data
reduction program may have generated and merge with the complete set,
discarding reflection with no Fobs (MNF) or with SigF=0.

In fact, if we consider a dataset is just a 3-dimensional array, or some
subset of it enclosing the reciprocal space asymmetric unit, I don't
see any reason we couldn't assign one universal P1 free-R set and
use it for every structure in whatever space group. By taking each
new dataset, merging with the universal Free-R, and discarding those
reflections not present in the new data, you would obtain a random
set for your structure. There could be nested (concentric?) free-R sets
with 10%, 5%, 2%, 1% free so that if you start out excluding 5% for a
low-res structure then get a high resolution dataset and want to exclude 2%,
you could be sure that all the 2% free reflections were also free in
your previous 5% set.

Thin or thick shells could be predefined. There may be problems when
it is desired to exclude reflections according to some twin law or NCS.

(just now read Nick Keep's post which expresses some similar ideas)
eab

On 06/04/2015 10:29 AM, Gerard Bricogne wrote:

Dear Graeme and other contributors to this thread,

  It seems to me that the how many is too many aspect of this
question, and the various culinary procedures that have been proposed
as answers, may have obscured another, much more fundamental issue,
namely: is it really the business of the data processing package to
assign FreeR flags?

  I would argue that it isn't. From the statistical viewpoint that
justifies the need for FreeR flags, these are pre-refinement entities
rather than post-processing ones. If one considers a single instance
of going from a dataset to a refined structure, then this distinction
may seem artificial. Consider, instead, the case of high-throughput
screening to detect fragment binding on a large number of crystals of
complexes between a given target protein (the apo) and a multitude
of small, weakly-binding fragments into solutions of which crystals of
the apo have been soaked.

  The model for the apo crystal structure comes from a refinement
against a dataset, using a certain set of FreeR flags. In order to
guard the detection of putative bound fragments against the evils of
model bias, it is very important to ensure that the refinement of each
complex against data collected on it does not treat as free any
reflections that were part of the working set in the refinement of the
apo structure. In other words, the free set for each complex must be
such that reflections that are also present in the apo dataset retain
the FreeR flag they had in that dataset. Any mixup, in the FreeR flags
for a complex, of the work vs. free status of the reflections also in
the apo would push Rwork up and Rfree down, invalidating their role as
indicators of quality of fit or of incipient overfitting.

  Great care must therefore be exercised, in the form of adequate
book-keeping and procedures for generating the FreeR flags in the mtz
file for each complex from that for the apo, to properly enforce this
inheritance of work vs. free status.

  In such a context there is a clear and crucial difference between
a post-processing entity and a pre-refinement one. FreeR flags belong
to the latter category. In fact, the creation of FreeR flags at the
end of the processing step can create a false perception, among people
doing ligand screening under pressure, that they cannot re-use the
FreeR flag information of the apo in refining their complexes, simply
because a new set has been created for each of them. This is clearly
to be avoided. Preserving the FreeR flags of the reflections that were
used in the refinement of the apo structure is one of the explicit
recommendations explicitly in the 2013 paper by Pozharski et al. (Acta
Cryst. D69, 150-167) - see section 1.1.3, p.152.

  Best practice in this area may therefore not be only a question
of numbers, but also of doing the appropriate thing in the appropriate
place. There are of course corner cases where e.g. substantial
unit-cell changes start to introduce some cross-talk between working
and free reflections, but the possibililty of such complications is no
argument to justify giving up on doing the right thing when the right
thing can be done.


  With best wishes,

   Gerard.

--
On Thu, Jun 04, 2015 at 08:30:57AM +, Graeme Winter wrote:

Hi Folks,

Many thanks for all of your comments - in keeping with the spirit of the BB
I have digested 

Re: [ccp4bb] crystal habit/morphology and the relationship to unit cell contents

[Edward A. Berry]

Is it possible to distinguish P64 from P62 without having basically solved the 
structure?

Given that it is P64,  is the +ive direction of the c axis arbitrary? A 
left-hand helix is left hand either way you point it, and the molecules in the 
asymmetric units could be pointing the opposite way.
Clearly the lattice is symmetric, so you could start out with either 
orientation and end up solving the structure. Phaser offers to try both 
enantiomers, but not to try with rotating the lattice to reverse the c axis in 
case you got that wrong.

I too would first guess that the truncated base is where it ran into the glass, 
and the flat tip
may be where it hit the air-water interface; the basic morphology being 
hexagonal bipyramid.
Unless there are dozens of crystals with the same habit.

eab

On 06/01/2015 03:45 PM, Roberts, Sue A - (suer) wrote:

Can't you break the ambiguity if there is significant anomalous signal? So, 
you'd need to have collected data from a protein crystal with SeMet, or a heavy 
atom, or near the S edge, and know the orientation of the crystal w.r.t. the 
direction of the unique crystal axis (for instance from face indexing.)

Sue

Dr. Sue A. Roberts
Dept. of Chemistry and Biochemistry
University of Arizona
1306 E. University Blvd,  Tucson, AZ 85721
Phone: 520 621 4168
s...@email.arizona.edu


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Phil 
Jeffrey
Sent: Monday, June 01, 2015 12:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystal habit/morphology and the relationship to unit 
cell contents

I would have thought that what the indexing routine defined as [001] vs [00-1] would be essentially 
random as one would obtain the equivalent indexing in 622 in both up and 
down alignment of the crystallographic a/b/c axes with respect to crystal morphology.

Phil Jeffrey
Princeton



On 6/1/15 1:44 PM, Scott Lovell wrote:

Hi Paul,

If you have access to diffractometer equipped with a 4-circle goniometer, you should be able to 
index the faces of the crystals.  All you need to do is collect some images to index the lattice 
and determine the orientation matrix.  Most instruments have software that allows one to then 
orient specific faces or crystallographic directions relative to various directions of the 
instrument (eg. camera, phi axis, direct beam, etc).  So after indexing, you could then orient the 
[001] direction of the crystal towards the camera to determine if this is the top or 
the base.  You can also determine the direction of the a/b axes [100] and [010] 
relative to the crystal and index the other faces.  If you can also measure the interfacial angles, 
this may help you to confirm the indices.

If you do this for a number of samples, is the top face always the [001] direction or 
is it the [00-1] direction for other crystals?  Assuming that you are growing these crystals by 
hanging drop, my guess is that the base is in contact with the coverslip during growth 
and you observe this half pyramid habit.  If you were to grow the crystals using the floating drop 
method, to prevent contact with the plate materials, would the crystals form a bipyramidal habit?  
Or do you see crystals in the current drop that have the same habit but are not in contact with the 
plate materials?

Scott


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Paul Paukstelis
Sent: Monday, June 01, 2015 11:21 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystal habit/morphology and the relationship to
unit cell contents

I'm interested in knowing how to figure out the relationship between the unit 
cell contents and the crystal habit in these crystals (small attachment, two 
roughly orthogonal views).

Space group is P64 (enantiomeric) , and you can clearly see the six-fold. The question becomes how 
to determine which direction the screw axis is going with respect to top and the 
base of the pyramidal crystals (right image) so I can gauge how/why the crystals grow 
this way based on the cell contents.

Thanks in advance.

--paul

Re: [ccp4bb] Two kinds of solvent-accessible-surface ?

[Edward A. Berry]

Thanks! guess I had it backwards.
eab

On 05/15/2015 03:19 PM, Yong Wang wrote:

Pymol should draw the molecular surface (or Connolly surface) by default.  If you want 
the solvent accessible surface, turn on the option solvent accessible under 
Setting/Surface.

Yong

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. 
Berry
Sent: Friday, May 15, 2015 3:08 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Two kinds of solvent-accessible-surface ?

If I remember correctly, there are two different ways to calculate a surface by 
rolling a ball over it, and i think that I want a program to calculate the 
non-conventional one.

As I understand, the ASA is defined as the surface traced out by the _center_ 
of the rolling sphere, i.e. one radius above the vdw surface. The justification 
being that an atom (i.e. it's center coordinates) can't get any closer to the 
surface than that.  The second type of surface is defined by the closest 
approach of the _surface_ of the rolling sphere, i.e. it would be the vdw 
radius of the (protein) but not descending into cracks between atoms where the 
rolling ball won't fit.

For making models of a multisubunit protein, and wanting to be able to assemble 
the separately-made subunits so that they make intermolecular vdw contacts, the 
second kind of surface would be desirable, as otherwise atoms won't be able to 
get their surfaces closer than twice the sphere radius. Is that an option that 
can be chosen in pymol or such?

Thanks,
eab

Re: [ccp4bb] [phenixbb] how to generate an isomorphous difference map

[Edward A. Berry]

OK. I guess I was thinking of subtracting 2fo-Fc maps, which would give 
essentially (Fo1-Fo2) coefficients. But coming out of phenix it will be at 
least 2mFo-DFc, with different m's and D's for the two datasets. Makes it 
rather more complicated.
eab

On 05/12/2015 11:58 AM, Philip Kiser wrote:

Good points. But also, the traditional isomorphous difference map is not as susceptible 
to model bias issues compared to a real space difference map (or a vector 
isomorphous difference map).

On Tue, May 12, 2015 at 11:37 AM, Edward A. Berry ber...@upstate.edu 
mailto:ber...@upstate.edu wrote:



On 05/12/2015 09:46 AM, Philip Kiser wrote:

crystallogrphy,

If you did everything correctly, you should be able to directly calculate the difference 
map in Coot from the MTZ file generated by PHENIX. You would need to open it with Open 
MTZ rather than Auto open MTZ and then select the appropriate map coefficients. 
Note that what Tim described (a real space difference map) is technically different from the 
traditional isomorphous difference map where the SF amplitudes of the two data sets are subtracted 
prior to the map calculation.

Philip


Should be the same, due to linear property of the Fourier transform, EXCEPT 
for possible scaling differences and treatment of missing data.  This latter 
could be significant, since presumably the traditional methods omits 
reflections unless present in both datasets, whereas in Tim's method each map 
will be made with all data available in that dataset. As a result, the largest 
coefficients in Tim's difference map might be the reflections which are absent 
in one map and present in the other.  But if both have good completeness, it  
may not matter.

eab



On Tue, May 12, 2015 at 9:24 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de 
mailto:t...@shelx.uni-ac.gwdg.de mailto:t...@shelx.uni-ac.gwdg.de 
mailto:t...@shelx.uni-ac.gwdg.de wrote:

 Dear crystallogrphy,

 you can open the PDB file with Coot, then use File - Open MTZ to 
read one
 mtz-file after the other into Coot. Coot will offer to generate 
the map
 coefficients from the previously loaded PDB file.

 Once you have both maps, you can go to Extensions - Maps - Make 
a Difference
 Map to create the difference map.

 In order to see both positive and negative density, you choose 
Extensions -
 Maps - Set Map is a Difference Map.

 Regards,
 Tim

 On Tue, May 12, 2015 at 08:43:45PM +0800, crystallogrphy wrote:
   Hi,
  
   I am trying to generate an isomorphous difference map with 
phenix GUI. I
   input reflection 1 and 2 with mtz format and a PDB model, I got 
an MTZ
   format map file. Because I want to load it to coot or pymol 
with negative
   and positive density, I need to transform this mtz map to ccp4 
map.
   However, FFT map cannot work.
   Does any know how can I get a ccp4 format maps with positive or 
negative
   density?
  
   Thanks!

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 --
 --
 Dr Tim Gruene
 Institut fuer anorganische Chemie
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 phone: +49 (0)551 39 22149 tel:%2B49%20%280%29551%2039%2022149 
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Re: [ccp4bb] Graphic for rotation about axis

[Edward A. Berry]

Thanks, these are great! I installed the ttf font on a windows box, and the 
symbols are available in MSWord and Photoshop (and presumably powerpoint and 
everything else). No more designing my own symbols in photoshop!
eab

On 05/08/2015 02:43 PM, Mooers, Blaine H.M. (HSC) wrote:

Hi Nick,

These symmetry element fonts at the IUCr website might be useful or at least 
inspirational:

http://www.iucr.org/resources/symmetry-font

Best regards,

Blaine

Blaine Mooers, Ph.D.
Assistant Professor
Director of the Laboratory of Biomolecular Structure and Function
Department of Biochemistry and Molecular Biology
University of Oklahoma Health Sciences Center
S.L. Young Biomedical Research Center Rm. 466

Shipping address:
975 NE 10th Street, BRC 466
Oklahoma City, OK 73104-5419

Letter address:
P.O. Box 26901, BRC 466
Oklahoma City, OK 73190

office: (405) 271-8300   lab: (405) 271-8313  fax:  (405) 271-3910
e-mail:  blaine-moo...@ouhsc.edu

Faculty webpage: 
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-

Small Angle Scattering webpage: 
http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-scattering-links-27aug2014.html?sfvrsn=0

X-ray lab webpage: 
http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Nicholas Keep 
[n.k...@mail.cryst.bbk.ac.uk]
Sent: Friday, May 08, 2015 9:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Graphic for rotation about axis

Where can you get hold of a graphic that indicates a rotation around the
horizontal or vertical axis
These occur in a lot of papers to indicate the relation between two
views but I don't seem to easily be able to google how to get hold of them
best wishes
Nick

--
Prof Nicholas H. Keep
Executive Dean of School of Science
Professor of Biomolecular Science
Crystallography, Institute for Structural and Molecular Biology,
Department of Biological Sciences
Birkbeck,  University of London,
Malet Street,
Bloomsbury
LONDON
WC1E 7HX

email n.k...@mail.cryst.bbk.ac.uk
Telephone 020-7631-6852  (Room G54a Office)
020-7631-6800  (Department Office)
Fax   020-7631-6803
If you want to access me in person you have to come to the crystallography 
entrance
and ring me or the department office from the internal phone by the door

Re: [ccp4bb] Alexander Rich passed away Monday April 27, 2015

[Edward A. Berry]

On 05/02/2015 12:23 PM, Imre Berger wrote:

Dear Edward -

Would you be so kind and explain why you went ahead to post that comment
about Alex Rich on CCP4, in a thread which announced the sad news of his
passing away?


Yes- I realized after posting it that it was inappropriate. If there is any way 
to remove a post, I will be glad to do so. In any case an apology is due.

As for the explanation, I did not intend it to be in any way derogatory.
I have never met Alex Rich, but Prof Sung-Hou Kim was my mentor in 
crystallography, and I have no doubt that their actions were completely 
honorable. As explained on the page I linked, it was all a misunderstanding 
based on poor communications between Kim and Rich, and rapid progress on the 
part of Kim that Rich was not aware of at the previous meeting. There was no 
evidence of actual misconduct on the part of Rich or Kim, as grudgingly 
acknowledged in the final letter from Cambridge. If only I had pointed that out 
in the email, instead of linking to that first accusatory message, it wouldn't 
have looked so bad. I had forgotten how inflammatory that first letter was!

I was thinking this followed in the lines of Bob Sweet's post, that Rich was a 
hard-driving man and maybe not afraid of stepping on some peoples toes in order 
to achieve his goals. I never meant to imply misconduct, although after reading 
back on my post I can see that interpretation.

My sincere apologies to the community and to the memory of professor Rich,
Ed Berry




I have checked your home page and your CV and it is not obvious to me at
all what motivation or stake you could possibly have.

Besides, knowing both Alex Rich and Aaron Klug  and having discussed
with them years ago, I think it is fair to say that only those two are
concerned with the issue, and one of them has - very sadly - just died.

In any case - in my view it is highly inappropriate indeed that you
placed those comments on CCP4.

Maybe you could be so kind and remove your contribution from the thread
- it would be greatly appreciated.

De mortuis nihil nisi bonum

Imre

--

Imre Berger PhD HDR
Professor of Biochemistry
Wellcome Trust Senior Investigator
Coordinator, EC FP7 ComplexINC project
The School of Biochemistry, University of Bristol UK
The European Molecular Biology Laboratory EMBL
imre.ber...@bristol.ac.uk
iber...@embl.fr