Re: [ccp4bb] Reprocess data with new resolution cutoff?

[Thomas Cleveland]

Hi everyone,

Thanks for all your input.  I will consider processing and depositing
the data out to 2.35 A, but refining only against data up to 2.55 A.

Just to give a little more information, and make sure what I'm doing
is reasonable, I am attaching below the table of statistics vs.
resolution from XSCALE.LP.  These data were collected from two
separate crystals and then merged.  The detector edge is at around
2.65 A, which is why completeness drops dramatically after this
point---I set XDS to integrate spots out to the corners of the
detector.

I have also attached my results from paired refinement.  My protein
has two domains, and there is an NMR model of each of the separate
domains.  The spacegroup is triclinic.  The structure was initially
solved by molecular replacement using the NMR structures, which finds
4 NCS copies of my protein (so 8 individual domains placed) in the
asymmetric unit.  Initial refinement was carried using default
parameters in Phenix, but using 10 macrocycles, NCS restraints, and
Cartesian simulated annealing, against the data cut at 2.35 A.  AFTER
this initial round of refinement (which took R/Rfree from 0.445/0.455
to 0.266/0.305), I carried out the paired refinements as described.

Since I haven't done this before, if anyone sees anything obviously
wrong that I have done, I would appreciate your comments.

Thanks,
Tom


Stats.pdf
Description: Adobe PDF document


Paired Refinement.pdf
Description: Adobe PDF document

[ccp4bb] R Factor

[Remie Fawaz-Touma]

Hi all,

I am trying to refine a structure in CCP4 and the final R factor is about 1% 
lower than the initial but for the first “task/cycle” within the same 
refinement job in CCP4, R factor starts off higher than the input data R factor.

I have a structure with multiple sugars attached, I did the first refinement 
after building the sugars with a lib file that worked well and the R factor did 
go down significantly but every time I try to refine again to lower it more, 
the above happens.

Any suggestions?

Thank you.

Remie

Re: [ccp4bb] odd phaser failure

[Airlie McCoy]

Dear Eleanor and Jie, 

I replied to Eleanor off-list to say that I can credit Isabel Uson with 
being the first to report this bug. The bug has been corrected for the 
recent phenix release. The phenix and ccp4 releases of phaser will be 
synchronised with the next ccp4 update, which is imminent.


Some ccp4bb-ers may not know that we have a reward for the first person to 
report any given bug. But you only get to find out what it is if you report 
a bug and also promise not to tell anyone what it is :-)


Airlie 


On May 20 2014, jie liu wrote:


Dear Eleanor

I've been getting this error for a while. Switching back to ccp4-6.3 
would solve the problem. I guess it's a bug of version 6.4.


Best

Jie
 - Original Message - 
 From: Eleanor Dodson 
 To: CCP4BB@JISCMAIL.AC.UK 
 Sent: Tuesday, May 20, 2014 5:26 AM

 Subject: [ccp4bb] odd phaser failure


 I should be sending thids to the phaser team I guess, but maybe there 
is an easy fix..


 This is the end of the log file..
 Sg I222 or I212121


Scoring 500 randomly sampled orientations and translations
Spreading calculation onto 2 threads.
Generating Statistics for TF SET #1 of 2
0%  100%
|===| DONE

Mean Score (Sigma):   -18.84   (17.72)


SET #1 of 2 TRIAL #1 of 245
---
Search Euler =   99.1   82.9  249.8, Ensemble = ensemble1

Doing Fast Translation Function FFT...
   Done

   Helix is perpendicular to two-fold: point exclusions applied

 
 $TEXT:Warning: $$ Baubles Markup $$
  
 
 
-
 UNHANDLED ERROR: cctbx Error: site_symmetry: min_distance_sym_equiv too 
large.
  
 
 
-

 $$


 
 EXIT STATUS: FAILURE
 

 CPU Time: 0 days 0 hrs 0 mins 28.27 secs ( 28.27 secs)
 Finished: Mon May 19 11:44:52 2014

 

Re: [ccp4bb] odd phaser failure

[jie liu]

Dear Airlie and Randy

Thank you! I have seen this error for quite some time and get it around by 
using ccp4-6.3 for Phaser. Will try to be the first to report next time :-)


All the best

Jie

- Original Message - 
From: "Airlie McCoy" 

To: "jie liu" 
Cc: 
Sent: Tuesday, May 20, 2014 12:08 PM
Subject: Re: [ccp4bb] odd phaser failure



Dear Eleanor and Jie,
I replied to Eleanor off-list to say that I can credit Isabel Uson with 
being the first to report this bug. The bug has been corrected for the 
recent phenix release. The phenix and ccp4 releases of phaser will be 
synchronised with the next ccp4 update, which is imminent.


Some ccp4bb-ers may not know that we have a reward for the first person to 
report any given bug. But you only get to find out what it is if you 
report a bug and also promise not to tell anyone what it is :-)


Airlie
On May 20 2014, jie liu wrote:


Dear Eleanor

I've been getting this error for a while. Switching back to ccp4-6.3 
would solve the problem. I guess it's a bug of version 6.4.


Best

Jie
 - Original Message - 
 From: Eleanor Dodson To: CCP4BB@JISCMAIL.AC.UK Sent: Tuesday, May 20, 
2014 5:26 AM

 Subject: [ccp4bb] odd phaser failure


 I should be sending thids to the phaser team I guess, but maybe there is 
an easy fix..


 This is the end of the log file..
 Sg I222 or I212121


Scoring 500 randomly sampled orientations and translations
Spreading calculation onto 2 threads.
Generating Statistics for TF SET #1 of 2
0%  100%
|===| 
DONE


Mean Score (Sigma):   -18.84   (17.72)


SET #1 of 2 TRIAL #1 of 245
---
Search Euler =   99.1   82.9  249.8, Ensemble = ensemble1

Doing Fast Translation Function FFT...
   Done

   Helix is perpendicular to two-fold: point exclusions applied

 
 $TEXT:Warning: $$ Baubles Markup $$
  
-
 UNHANDLED ERROR: cctbx Error: site_symmetry: min_distance_sym_equiv too 
large.

  
-
 $$


 
 EXIT STATUS: FAILURE
 

 CPU Time: 0 days 0 hrs 0 mins 28.27 secs ( 28.27 secs)
 Finished: Mon May 19 11:44:52 2014

 

Re: [ccp4bb] odd phaser failure

[Randy Read]

Hi,

I guess Airlie only replied to Eleanor off-line.  This is a problem that Isabel 
Uson had already reported to us.  It’s fixed in the current version of Phaser, 
which is in the pipeline to be released through the CCP4 update mechanism.

Best wishes,

Randy Read

On 20 May 2014, at 16:53, jie liu  wrote:

> Dear Eleanor
>  
> I've been getting this error for a while. Switching back to ccp4-6.3 would 
> solve the problem. I guess it's a bug of version 6.4.
>  
> Best
>  
> Jie
> - Original Message -
> From: Eleanor Dodson
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Tuesday, May 20, 2014 5:26 AM
> Subject: [ccp4bb] odd phaser failure
> 
> I should be sending thids to the phaser team I guess, but maybe there is an 
> easy fix..
> 
> This is the end of the log file..
> Sg I222 or I212121
> 
> 
>Scoring 500 randomly sampled orientations and translations
>Spreading calculation onto 2 threads.
>Generating Statistics for TF SET #1 of 2
>0%  100%
>|===| DONE
> 
>Mean Score (Sigma):   -18.84   (17.72)
> 
> 
>SET #1 of 2 TRIAL #1 of 245
>---
>Search Euler =   99.1   82.9  249.8, Ensemble = ensemble1
> 
>Doing Fast Translation Function FFT...
>   Done
> 
>   Helix is perpendicular to two-fold: point exclusions applied
> 
> 
> $TEXT:Warning: $$ Baubles Markup $$
> -
> UNHANDLED ERROR: cctbx Error: site_symmetry: min_distance_sym_equiv too large.
> -
> $$
> 
> 
> 
> EXIT STATUS: FAILURE
> 
> 
> CPU Time: 0 days 0 hrs 0 mins 28.27 secs ( 28.27 secs)
> Finished: Mon May 19 11:44:52 2014
> 
> 

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

Re: [ccp4bb] odd phaser failure

[jie liu]

Dear Eleanor

I've been getting this error for a while. Switching back to ccp4-6.3 would 
solve the problem. I guess it's a bug of version 6.4.

Best

Jie
  - Original Message - 
  From: Eleanor Dodson 
  To: CCP4BB@JISCMAIL.AC.UK 
  Sent: Tuesday, May 20, 2014 5:26 AM
  Subject: [ccp4bb] odd phaser failure


  I should be sending thids to the phaser team I guess, but maybe there is an 
easy fix..

  This is the end of the log file..
  Sg I222 or I212121


 Scoring 500 randomly sampled orientations and translations
 Spreading calculation onto 2 threads.
 Generating Statistics for TF SET #1 of 2
 0%  100%
 |===| DONE

 Mean Score (Sigma):   -18.84   (17.72)


 SET #1 of 2 TRIAL #1 of 245
 ---
 Search Euler =   99.1   82.9  249.8, Ensemble = ensemble1

 Doing Fast Translation Function FFT...
Done

Helix is perpendicular to two-fold: point exclusions applied

  
  $TEXT:Warning: $$ Baubles Markup $$
  
-
  UNHANDLED ERROR: cctbx Error: site_symmetry: min_distance_sym_equiv too large.
  
-
  $$


  
  EXIT STATUS: FAILURE
  

  CPU Time: 0 days 0 hrs 0 mins 28.27 secs ( 28.27 secs)
  Finished: Mon May 19 11:44:52 2014

  

Re: [ccp4bb] [ccp4bb] map manipulation questions

[Randy Read]

Oh no, I’ve been meaning to update that page for a while!  It’s much easier to 
cut out density now than it was when I wrote that.  In CCP4, Kevin Cowtan has 
the program cmapcut, and in Phenix Tom Terwilliger has the program 
phenix.cut_out_density.  For most purposes, I think you’ll find those programs 
easier to use.

Best wishes,

Randy Read

On 20 May 2014, at 14:52, Edward A. Berry  wrote:

> But, if you convert to structure factors and recalculate the map in a 
> different cell,
> the features will be "stretched" to fill the cell, which I take it is the 
> original problem.
> 
> I found Kleywegt's RAVE package very convenient for doing this,
> but i believe the functionality is now available in
> ccp4 mapmask and maprot programs.
> 
> The Phaser Wiki has a page with instructions
> for cutting out electron density within a mask,
> and putting it in a large P1 cell for use as a molecular
> replacement model. Perhaps you could modify that to achieve
> your aims.
> 
> http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model
> eab
> 
> On 05/20/2014 03:07 AM, herman.schreu...@sanofi.com wrote:
>> Dear Niu,
>> 
>> Provided you have a complete asymmetric unit (unit cell in P1), you could 
>> also convert this map to
>> structure factors and manipulate those, e.g. with sftools. To calculate 
>> structure factors you could use
>> sfall and also clipper has utilities to convert a map to structure factors.
>> 
>> Best,
>> 
>> Herman
>> 
>> *Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von 
>> *Niu Tou
>> *Gesendet:* Montag, 19. Mai 2014 23:25
>> *An:* CCP4BB@JISCMAIL.AC.UK
>> *Betreff:* [ccp4bb] map manipulation questions
>> 
>> Dear All,
>> 
>> I have a ccp4 format map file in P1 spacegroup, I would like to manipulate 
>> it in several ways:
>> 
>> 1. enlarge the cell dimension . When I tried "CELL" keyword in MAPMAN, the 
>> density scaled up together
>> with the cell dimension. Does anybody know how to do it without changing the 
>> density?
>> 
>> 2. Change the space group to P2.
>> 
>> 3. Move the density away from its original place, i.e. apply a translocation 
>> vector to it.
>> 
>> Does anybody know the answers? Thanks in advance!
>> 
>> Regards,
>> 
>> Niu
>> 

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

Re: [ccp4bb] Xia2 / XDS issues

[Antony Oliver]

Dear all,

Firstly - many thanks to Kay Diederichs and Graeme Winter.

The “memory” error was in fact due to certain diffraction images having very 
few (weak), if any spots - essentially they were ‘blank’.
By excluding these images, and with a combination of Xia2, plus XDS + Aimless, 
I was finally able to satisfactorily process the data.

FYI: The resultant mtz extends to ~ 3.6 Angstrom, with a CC(1/2) of 0.633.
Although the resolution isn’t the greatest, 8-fold NCS makes the resultant maps 
rather nice.

A great many thanks to others who offered useful tips and advice.

A slightly less tired Antony.

- - - - - - - - - - - - - - - - - -
Dr Antony W Oliver
Senior Research Fellow
CR-UK DNA Repair Enzymes Group
Genome Damage and Stability Centre
Science Park Road
University of Sussex
Falmer, Brighton, BN1 9RQ
- - - - - - - - - - - - - - - - - -
email: antony.oli...@sussex.ac.uk

tel (office): +44 (0)1273 678349
tel (lab): +44 (0)1273 677512

http://www.sussex.ac.uk/lifesci/oliverlab
http://tinyurl.com/aw-oliver
- - - - - - - - - - - - - - - - - -

Re: [ccp4bb] AW: [ccp4bb] map manipulation questions

[Gerard DVD Kleywegt]

MAPMAN contains several related options, e.g. CEll, SPacegroup, TRanslate and 
GTranslate, PAste (see the manual for caveats - 
http://xray.bmc.uu.se/usf/mapman_man.html ). Saving as an ASCII file and 
editing may also work, if you know what you are doing.


--Gerard





On Tue, 20 May 2014, Edward A. Berry wrote:

But, if you convert to structure factors and recalculate the map in a 
different cell,
the features will be "stretched" to fill the cell, which I take it is the 
original problem.


I found Kleywegt's RAVE package very convenient for doing this,
but i believe the functionality is now available in
ccp4 mapmask and maprot programs.

The Phaser Wiki has a page with instructions
for cutting out electron density within a mask,
and putting it in a large P1 cell for use as a molecular
replacement model. Perhaps you could modify that to achieve
your aims.

http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model
eab

On 05/20/2014 03:07 AM, herman.schreu...@sanofi.com wrote:

Dear Niu,

Provided you have a complete asymmetric unit (unit cell in P1), you could 
also convert this map to
structure factors and manipulate those, e.g. with sftools. To calculate 
structure factors you could use

sfall and also clipper has utilities to convert a map to structure factors.

Best,

Herman

*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von 
*Niu Tou

*Gesendet:* Montag, 19. Mai 2014 23:25
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [ccp4bb] map manipulation questions

Dear All,

I have a ccp4 format map file in P1 spacegroup, I would like to manipulate 
it in several ways:


1. enlarge the cell dimension . When I tried "CELL" keyword in MAPMAN, the 
density scaled up together
with the cell dimension. Does anybody know how to do it without changing 
the density?


2. Change the space group to P2.

3. Move the density away from its original place, i.e. apply a 
translocation vector to it.


Does anybody know the answers? Thanks in advance!

Regards,

Niu






Best wishes,

--Gerard

**
   Gerard J. Kleywegt

  http://xray.bmc.uu.se/gerard   mailto:ger...@xray.bmc.uu.se
**
   The opinions in this message are fictional.  Any similarity
   to actual opinions, living or dead, is purely coincidental.
**
   Little known gastromathematical curiosity: let "z" be the
   radius and "a" the thickness of a pizza. Then the volume
of that pizza is equal to pi*z*z*a !
**

Re: [ccp4bb] AW: [ccp4bb] map manipulation questions

[Edward A. Berry]

But, if you convert to structure factors and recalculate the map in a different 
cell,
the features will be "stretched" to fill the cell, which I take it is the 
original problem.

I found Kleywegt's RAVE package very convenient for doing this,
but i believe the functionality is now available in
ccp4 mapmask and maprot programs.

The Phaser Wiki has a page with instructions
for cutting out electron density within a mask,
and putting it in a large P1 cell for use as a molecular
replacement model. Perhaps you could modify that to achieve
your aims.

http://www.phaser.cimr.cam.ac.uk/index.php/Using_Electron_Density_as_a_Model
eab

On 05/20/2014 03:07 AM, herman.schreu...@sanofi.com wrote:

Dear Niu,

Provided you have a complete asymmetric unit (unit cell in P1), you could also 
convert this map to
structure factors and manipulate those, e.g. with sftools. To calculate 
structure factors you could use
sfall and also clipper has utilities to convert a map to structure factors.

Best,

Herman

*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Niu 
Tou
*Gesendet:* Montag, 19. Mai 2014 23:25
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [ccp4bb] map manipulation questions

Dear All,

I have a ccp4 format map file in P1 spacegroup, I would like to manipulate it 
in several ways:

1. enlarge the cell dimension . When I tried "CELL" keyword in MAPMAN, the 
density scaled up together
with the cell dimension. Does anybody know how to do it without changing the 
density?

2. Change the space group to P2.

3. Move the density away from its original place, i.e. apply a translocation 
vector to it.

Does anybody know the answers? Thanks in advance!

Regards,

Niu

[ccp4bb] Two postdoctoral positions available at UCL/Magnus Life Science

[Yelland, Tamas]

Hi ccp4bb

Two postdoctoral positions in X-ray crystallography/drug discovery are 
currently available at University College London/Magnus Life Science.  Please 
see link for further details.

http://www.magnuslifescience.co.uk/careers/

Best

[ccp4bb] odd phaser failure

[Eleanor Dodson]

I should be sending thids to the phaser team I guess, but maybe there is an
easy fix..

This is the end of the log file..
Sg I222 or I212121


   Scoring 500 randomly sampled orientations and translations
   Spreading calculation onto 2 threads.
   Generating Statistics for TF SET #1 of 2
   0%  100%
   |===| DONE

   Mean Score (Sigma):   -18.84   (17.72)


   SET #1 of 2 TRIAL #1 of 245
   ---
   Search Euler =   99.1   82.9  249.8, Ensemble = ensemble1

   Doing Fast Translation Function FFT...
  Done

  Helix is perpendicular to two-fold: point exclusions applied


$TEXT:Warning: $$ Baubles Markup $$
-
UNHANDLED ERROR: cctbx Error: site_symmetry: min_distance_sym_equiv too
large.
-
$$



EXIT STATUS: FAILURE


CPU Time: 0 days 0 hrs 0 mins 28.27 secs ( 28.27 secs)
Finished: Mon May 19 11:44:52 2014

Re: [ccp4bb] [off topic] reference request / thanks for your help. Already got the references.

[JinSoo.Bae]

2014. 5. 20. 오후 4:25에 "JinSoo.Bae" 님이 작성:

> Dear all,
>
> Is there any one who have 2 references as below?
>
> Processing of X-ray diffraction data collected in oscillation mode.
> Methods Enzymol. 276: 307-326
>
>  PROCHECK - a program to check the stereochemical quality of protein
> structures. J. App. Cryst. 26: 283-291
>
> please kindly send to me.
>
> thanks in advance!
>
>
> Best regard,
> Jin Soo Bae
> 790-784
> room 204, Dept of Life Science,
> POSTECH, san31, Hyoja-dong,
> Nam-gu, Pohang, Gyungbuk, Korea
> tel:82-54-279-8627 fax:82-54-279-8111
>

Re: [ccp4bb] Reprocess data with new resolution cutoff?

[Kay Diederichs]

Hi Thomas,

strictly speaking, there is no need to re-process the data (because it wouldn't 
change the data anyway). It may even be that the data beyond 2.55A contain some 
signal (CC1/2 should tell you about that), which future refinement programs can 
use - so you could deposit at the RCSB all data out to 2.35A (if that is where 
CC1/2 becomes insignificant), and future PDB_REDO calculations could make use 
of that.

What most people still do, however, is that they make an attempt (for the 
infamous Table 1 ...) to match the high-resolution cutoff of the data 
processing to the high-resolution cutoff of refinement. I say "attempt" because 
the width of the highest-resolution shell (of the data processing and the 
refinement program) in most cases then still does _not_ match. And AFAIK there 
is no formal requirement of any journal to have the same cutoff. But having the 
same cutoff is based on the old and flawed assumption that Rmerge in the 
highest resolution shell tells you something about the "quality" of the data 
being used for refinement. 

What I personally do is to adjust the cutoff in data processing such that CC1/2 
in the highest-resolution shell is still significant at the 1-in-1000 level 
(this information is available from the XDS output). I stick with this dataset 
which has all the information about the crystal and is future-proof in the 
sense mentioned above. I then do paired refinement to find out what the best 
high-resolution cutoff is, and I find that this depends to some extent on the 
refinement program and the quality of the model. So I might end up with a 
slightly lower high-resolution cutoff in refinement than I do in data 
processing, but I (and usually the reviewers!) don't consider this a real 
problem.

hope this helps,

Kay



On Mon, 19 May 2014 23:07:54 -0400, Thomas Cleveland 
 wrote:

>Hi all,
>
>This is a basic question and I'm sure the answer is widely known, but
>I'm having trouble finding it.
>
>I'm working on my first structure.  I have a dataset that I processed
>in XDS with a resolution cutoff of 2.35 A, although the data are
>extremely weak-to-nonexistent at that resolution limit.  After
>successful molecular replacement and initial refinement, I then
>performed "paired refinements" against this dataset cut to various
>resolutions (2.95 A, 2.85 A, 2.75 A, etc).  Based on the improvement
>in R/Rfree seen between successive pairs, it appears that the data
>should be cut at around 2.55 A.
>
>Here is my question: as I proceed with refinement (I'm currently using
>Phenix), should I now simply set "2.55 A" as the resolution limit in
>Phenix?  Or should I go back to XDS and actually reprocess the data
>with the new limit (2.55 A instead of 2.35 A)?
>
>Thanks,
>Tom

Re: [ccp4bb] Xia2 / XDS issues

[Kay Diederichs]

Dear Tony,

I renamed your LP_01.tmp to INTEGRATE.LP and ran XDSGUI - this shows nicely 
that after frame 150 things "fall over".

So pls try the recommendations in the "Difficult datasets" article of XDSwiki.

Normally you should not need to run two processing jobs; just prevent 
refinement from "running away".

best,

Kay


On Mon, 19 May 2014 16:31:19 +, Antony Oliver  
wrote:

>Dear CCP4-ers.
>
>Many apologies for the previous post (with attachments).
>
>However, it is true -  somewhat crappy data, that I can�t seem to reprocess 
>with XDS / xia2.
>
>Tony.
>
>HI Graeme,
>
>Please find below, what I think it is that you need?  Files attached, plus 
>relevant text clips,.
>
>NB: everything seems to fail at the integration step.
>
>NB2: This is quite crappy data�.(!)
>
>Tony.
>
>- - - - - - - - - - - - - - - - - -
>Dr Antony W Oliver
>Senior Research Fellow
>CR-UK DNA Repair Enzymes Group
>Genome Damage and Stability Centre
>Science Park Road
>University of Sussex
>Falmer, Brighton, BN1 9RQ
>- - - - - - - - - - - - - - - - - -
>email: antony.oli...@sussex.ac.uk
>
>tel (office): +44 (0)1273 678349
>tel (lab): +44 (0)1273 677512
>
>http://www.sussex.ac.uk/lifesci/oliverlab
>http://tinyurl.com/aw-oliver
>- - - - - - - - - - - - - - - - - -
>
>
>

Re: [ccp4bb] data processing with XDS

[Kay Diederichs]

Dear Almudena,

if you follow the recommendations given in the "Difficult datasets" article on 
XDSwiki, your problem would have a good chance to be solved.

best wishes,

Kay

On Mon, 19 May 2014 18:18:46 +0200, Almudena Ponce Salvatierra 
 wrote:

>Dear all,
>
>I am looking for some suggestions here. I have lately collected some
>datasets but the spots are very very weak... it is very difficult to
>process them. At times it looks like XDS is stalled or at times it just
>says that it can not interpret the lattice parameters... also while running
>integrate I have such a message after each range of images:
>
>!!! WARNING !!! REFINEMENT DID NOT CONVERGE
> LAST CORRECTION SHIFT WAS   9.1E-03 (should be <  1.0E-03)
>
>I guess this is not good at all. And I wonder what to do? Is there any info
>that I can get out from these data at all? or rather not?
>
>I wonder if the problem is to find the spots, I have tried with HKL2000 and
>it can't even find enough of them for indexing.
>
>Any ideas are welcome.
>
>Best wishes,
>
>Almudena.
>
>-- 
>Almudena Ponce-Salvatierra
>Macromolecular crystallography and Nucleic acid chemistry
>Max Planck Institute for Biophysical Chemistry
>Am Fassberg 11 37077 Göttingen
>Germany
>

Re: [ccp4bb] Xia2 / XDS issues

[Graeme Winter]

Dear Tony,

OK, clearly Tim's suggestion is spot on, to remove the offending images
from processing (this is easily done by splitting your input xinfo or the
automatically generated one, I will copy an example below)

I suspect that the "memory error" results from

 IMAGE IER  SCALE NBKG NOVL NEWALD NSTRONG  NREJ  SIGMAB  SIGMAR
   189   0  1.038  90884180  14057   2 0  0.0069  3.
   190   0  1.037  90870920  14088   0 0  0.  0.
   191   0  1.039  90879820  14075   2 0  0.0260  0.9701

where you have a fairly large mosaic spread on one image and 0.0 on the
next  - with the way XDS works with processing images this can't be good.
It is clearly an artefact of the complete absence of strong spots however
good to have some kind of reasoning for this.

Best wishes, Graeme

Splitting xinfo files:

If you ran with -3dii you should have a SPOT.XDS file in the index
directory, which has strong spots from throughout the data set. If you plot
with e.g. gnuplot (a bit old school, I should automate this) the SPOT.XDS
file as

Terminal type set to 'x11'
gnuplot> plot 'SPOT.XDS' using 3:4 with dots

you should get an idea of which images were very poor. Keep these in mind.

Automatic.xinfo will include something like:

BEGIN SWEEP SWEEP1
WAVELENGTH NATIVE
DIRECTORY /data1/gw56/demo
IMAGE insulin_1_001.img
START_END 1 45
BEAM  94.34  94.50
END SWEEP SWEEP1

what you will want to do is split this into 1A and 1B (say) e.g.

BEGIN SWEEP SWEEP1A
WAVELENGTH NATIVE
DIRECTORY /data1/gw56/demo
IMAGE insulin_1_001.img
START_END 1 22
BEAM  94.34  94.50
END SWEEP SWEEP1A

BEGIN SWEEP SWEEP1B
WAVELENGTH NATIVE
DIRECTORY /data1/gw56/demo
IMAGE insulin_1_001.img
START_END 26 45
BEAM  94.34  94.50
END SWEEP SWEEP1B

which will ignore images 23, 24, 25











On 19 May 2014 17:09, Antony Oliver  wrote:

>  HI Graeme,
>
>  Please find below, what I think it is that you need?  Files attached,
> plus relevant text clips,.
>
>  NB: everything seems to fail at the integration step.
>
>  NB2: This is quite crappy data….(!)
>
>  Tony.
>
>  >XDS.INP
>
>   JOB=INTEGRATE
>  MAXIMUM_NUMBER_OF_PROCESSORS=8
>  TEST=2
>  DELPHI=5.0
>  REFINE(INTEGRATE)=ORIENTATION CELL BEAM DISTANCE
>  DETECTOR=ADSC MINIMUM_VALID_PIXEL_VALUE=1 OVERLOAD=65000
>  DIRECTION_OF_DETECTOR_X-AXIS=1.00 0.00 0.00
>  DIRECTION_OF_DETECTOR_Y-AXIS=0.00 1.00 -0.00
>  TRUSTED_REGION=0.0 1.41
>  NX=3072 NY=3072 QX=0.102600 QY=0.102600
>  DETECTOR_DISTANCE=456.750
>  OSCILLATION_RANGE=2.00
>  X-RAY_WAVELENGTH=0.979500
>  ROTATION_AXIS= 1.000 0.000 0.000
>  INCIDENT_BEAM_DIRECTION=0.0 0.0 1.0
>  FRACTION_OF_POLARIZATION=0.99
>  POLARIZATION_PLANE_NORMAL=0.0 1.0 0.0
>  AIR=0.001
> NAME_TEMPLATE_OF_DATA_FRAMES=/Volumes/Data/diamond/I02_0211/Mohan/TNK1/xtal1/xtal1_M1S1_4_???.img
>  DATA_RANGE=1 200
>
>  >9_xds_par_INTEGRATE.log
>
>   
> **
>   PROCESSING OF IMAGES  189 ... 191
>  
> **
>
>   USING   3 PROCESSORS
>
>   *** DEFINITION OF SYMBOLS ***
>   IER = ERROR CODE AFTER ACCESSING DATA IMAGE
>0: NO ERROR
>   -1: CANNOT OPEN OR READ IMAGE FILE
>   -3: WRONG DATA FORMAT
>   SCALE   = SCALING FACTOR FOR THIS DATA IMAGE
>   NBKG= NUMBER OF BACKGROUND PIXELS ON DATA IMAGE
>   NOVL= NUMBER OF OVERLOADED REFLECTIONS ON DATA IMAGE
>   NEWALD  = NUMBER OF REFLECTIONS CLOSE TO THE EWALD SPHERE
>   NSTRONG = NUMBER OF STRONG REFLECTIONS ON DATA IMAGE
>   NREJ= NUMBER OF UNEXPECTED REFLECTIONS
>   SIGMAB  = BEAM_DIVERGENCE_E.S.D.=SIGMAB
>   SIGMAR  = REFLECTING_RANGE_E.S.D.=SIGMAR (MOSAICITY)
>
>   IMAGE IER  SCALE NBKG NOVL NEWALD NSTRONG  NREJ  SIGMAB  SIGMAR
> 189   0  1.038  90884180  14057   2 0  0.0069  3.
> 190   0  1.037  90870920  14088   0 0  0.  0.
> 191   0  1.039  90879820  14075   2 0  0.0260  0.9701
>
>   !!! WARNING !!! UNABLE TO CARRY OUT REFINEMENT. RETURN CODE  IER=
> 0
>   XDS WILL CONTINUE WITH ORIGINAL DIFFRACTION PARAMETER VALUES.
>
>   !!! ERROR !!! CANNOT ALLOCATE MEMORY
> YOU COULD RERUN THIS STEP WITH SMALLER VALUES FOR THE
> PARAMETERS
> NUMBER_OF_PROFILE_GRID_POINTS_ALONG_ALPHA/BETA=
> NUMBER_OF_PROFILE_GRID_POINTS_ALONG_GAMMA=
>  # command line:
>  # xds_par
>
>

[ccp4bb] AW: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular Replacement

[Herman . Schreuder]

Dear Matt,

I am afraid you are in a difficult position. With poor 4Å data, you need a 
convincing MR solution to be sure that the solution is correct.
The large number of molecules in the a.s.u. is probably causing the poor 
diffraction.

Things I would do are:
If you have more crystals, try them all. One of them may diffract better or 
have a better behaved crystal packing.
If you don’t have more crystals, grow more and try variations and additives.
It probably won’t work because your protein is monomeric, but try to construct 
a model of a dimer or tetramer, based on the crystal packing of suitable search 
models. I am afraid that this is not going to work with NMR models.
Trim your model, e.g. remove potentially flexible surface loops and large side 
chains on the surface.
Try a different construct. A slightly longer or shorter construct may give a 
radically different crystal packing.

Good luck!
Herman



Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Matthew 
Bratkowski
Gesendet: Montag, 19. Mai 2014 22:48
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] Issue with Molecules per Asymmetric Unit for Molecular 
Replacement

Hello all,

Thank you for you suggestions.  I took a look at the crystal packing for the 
solution with one molecule per asu, and the next closest molecule is 50 
angstroms away, suggesting that this is not likely the correct solution.  I 
have also tried MR with a number of different molecules per asu.  In some cases 
I get better packing, but I have not yet gotten a solution that looks to refine 
well.  In addition to my previous information, I would like to add the 
following:

1. The resolution of my data is not particular great.  I get ~4 A resolution at 
best and spots are rather weak even with almost no beam attenuation.
2. The search model that gives the best solution (in terms of contrast score in 
MolRep) is an NMR structure.  I have heard that MR with NMR structures can 
possibly give false solutions.  An alternate crystal structure that I tried 
using gave much poorer contrast socres overall, regardless of the number of 
molecules in the asu to search for.

If anyone has any additonal suggestion, I would appreciate them.

Thanks,
Matt

On Fri, May 16, 2014 at 8:46 AM, R. M. Garavito 
mailto:rmgarav...@gmail.com>> wrote:
Matt,

In addition to the suggestions of the others, have you done a simple self 
rotation function?  It can tell you quite a bit about how things are packed and 
give you strict criteria for choosing one solution over another.  As Roger 
said, choosing an even number of monomers in the ASU is a good strategy, 
particularly if the self rotation function shows NCS 2-folds.

Also, a calculated Matthews coefficient is NEVER correct, it is a mere 
guideline; it only has validity for any particularly crystal form AFTER the 
fact.  Let the number of monomers in the ASU vary from 6-10; I have had MR 
cases that have had as little as 40% solvent to 70% solvent, where the 
calculated Matthews coefficient was quite "wrong" (i.e., the most common value 
observed in OTHER crystals).   Two things to watch out for are:

(1) An odd number of monomers in the ASU.  I have had 1 1/2 dimers in an ASU 
(the 1/2 dimer is paired with another in a neighboring ASU).  It is sometimes 
confusing to people and occasionally difficult to solve with some MR programs 
due to clashes.

(2) Translation symmetry, which still can confuse some programs (but they are 
are getting better at detecting it).

Finally, as Herman pointed out, look at the packing of any solution you are 
considering.  It is surprising how a correct solution "looks" correct: nice 
intermolecular contacts and a pleasing distribution of mass throughout the unit 
cell (meaning expand out to at least a unit cell volume, which is easy in 
Pymol).  Any unexplained gaps (meaning not caused by a missing domain) should 
be viewed critically.

Regards and Good Luck,

Michael


R. Michael Garavito, Ph.D.
Professor of Biochemistry & Molecular Biology
603 Wilson Rd., Rm. 513
Michigan State University
East Lansing, MI 48824-1319
Office:  (517) 355-9724 Lab:  (517) 
353-9125
FAX:  (517) 353-9334Email:  
rmgarav...@gmail.com




On May 15, 2014, at 6:50 PM, Matthew Bratkowski 
mailto:mab...@cornell.edu>> wrote:

Hello all,


I am working on the structure of a small protein in space group P212121.  The 
protein is monomeric in solution based on gel filtration analysis.  The 
Matthews Coefficeint program indicates that 9-10 molecules per asymmetric unit 
results in ~50% solvent content, while 1 molecule per asymmetric unit results 
in ~95% solvent.

 I tried molecular replacement with a search model which is essentially 
identical in sequence to my protein, and searched for 9 or 10 molecules/asu.  
Using MolRep with 9 or 10 molecules/asu, I get p

Re: [ccp4bb] Reprocess data with new resolution cutoff?

[Tim Gruene]

Dear Thomas,

I would object to Zbyszek's recommendation. If I calculated correctly
(2.55/2.35)^3, roughly 20% of your reflections were integrated as noise.
Due to how profile fitting and scaling work this may affect your overall
data quality. Also your parameters may be partially refined against
noisy data.

Take a look at the scaling plots vs. resolution and compare before and
after.

I would certainly reprocess the data to your final resolution. Anyhow,
it takes probably less than an hour to reprocess.

Would you tell us the CC(1/2) in the highest resolution shell now and
when you reprocess to 2.55A?

Best,
Tim

On 05/20/2014 05:07 AM, Thomas Cleveland wrote:
> Hi all,
> 
> This is a basic question and I'm sure the answer is widely known, but
> I'm having trouble finding it.
> 
> I'm working on my first structure.  I have a dataset that I processed
> in XDS with a resolution cutoff of 2.35 A, although the data are
> extremely weak-to-nonexistent at that resolution limit.  After
> successful molecular replacement and initial refinement, I then
> performed "paired refinements" against this dataset cut to various
> resolutions (2.95 A, 2.85 A, 2.75 A, etc).  Based on the improvement
> in R/Rfree seen between successive pairs, it appears that the data
> should be cut at around 2.55 A.
> 
> Here is my question: as I proceed with refinement (I'm currently using
> Phenix), should I now simply set "2.55 A" as the resolution limit in
> Phenix?  Or should I go back to XDS and actually reprocess the data
> with the new limit (2.55 A instead of 2.35 A)?
> 
> Thanks,
> Tom
> 

-- 
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A



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[ccp4bb] [off topic] reference request

[JinSoo.Bae]

Dear all,

Is there any one who have 2 references as below?

Processing of X-ray diffraction data collected in oscillation mode. Methods
Enzymol. 276: 307-326

 PROCHECK - a program to check the stereochemical quality of protein
structures. J. App. Cryst. 26: 283-291

please kindly send to me.

thanks in advance!


Best regard,
Jin Soo Bae
790-784
room 204, Dept of Life Science,
POSTECH, san31, Hyoja-dong,
Nam-gu, Pohang, Gyungbuk, Korea
tel:82-54-279-8627 fax:82-54-279-8111

[ccp4bb] AW: [ccp4bb] map manipulation questions

[Herman . Schreuder]

Dear Niu,

Provided you have a complete asymmetric unit (unit cell in P1), you could also 
convert this map to structure factors and manipulate those, e.g. with sftools. 
To calculate structure factors you could use sfall and also clipper has 
utilities to convert a map to structure factors.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Niu Tou
Gesendet: Montag, 19. Mai 2014 23:25
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] map manipulation questions

Dear All,

I have a ccp4 format map file in P1 spacegroup, I would like to manipulate it 
in several ways:

1. enlarge the cell dimension . When I tried "CELL" keyword in MAPMAN, the 
density scaled up together with the cell dimension. Does anybody know how to do 
it without changing the density?

2. Change the space group to P2.

3. Move the density away from its original place, i.e. apply a translocation 
vector to it.

Does anybody know the answers? Thanks in advance!

Regards,
Niu