Re: [ccp4bb] Tricks to solubilize protein
Hi The low pH and its interaction with PIP1 can be conflicting. The low pH can modify the interaction site. Nevertheless, one good way to study protein stability is thermal shift assay. You can read this publication too : PubMed ID 16604423. (Rapid determination of protein solubility and stability conditions for NMR studies using incomplete factorial design). Michel.
Re: [ccp4bb] question about processing data
I think the answer to your question depends on why the data is incomplete. James On Mar 17, 2008, at 3:06 AM, Melody Lin wrote: Hi all, I have always been wondering... for a data set diffracting to say 2.15 Angstrom but in the highest resolution shell (2.25-2.15) the completeness is 74%, should I use merge all the data and call it a 2.15 A dataset or I should cut the data set to say 2.25 A where the highest resolution shell has better completeness (85%)? What is an acceptable completeness value for the highest resolution shell? Thank you. Best, Melody -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
[ccp4bb] question about processing data
Hi all, I have always been wondering... for a data set diffracting to say 2.15Angstrom but in the highest resolution shell ( 2.25-2.15) the completeness is 74%, should I use merge all the data and call it a 2.15 A dataset or I should cut the data set to say 2.25 A where the highest resolution shell has better completeness (85%)? What is an acceptable completeness value for the highest resolution shell? Thank you. Best, Melody
Re: [ccp4bb] question about processing data
Hi Melody, There was a nice discussion in this year's ccp4 study weekend. In general, one needs to consider several factors.. If you were at 3A, or low symmetry, you would of course try to get the maximum out of it, on the other hand, there are requirements for experimental phasing.. in general, judge it from: 1. Completeness 2. Redundancy 3. I / Sigma 4. R merge statistics Not just one of them. If you are pushing it too far, you will see the effect in later refinement step.. With 74% completeness, how does the other parameters look like? HTH, Partha On Mon, Mar 17, 2008 at 10:06 AM, Melody Lin [EMAIL PROTECTED] wrote: Hi all, I have always been wondering... for a data set diffracting to say 2.15 Angstrom but in the highest resolution shell (2.25-2.15) the completeness is 74%, should I use merge all the data and call it a 2.15 A dataset or I should cut the data set to say 2.25 A where the highest resolution shell has better completeness (85%)? What is an acceptable completeness value for the highest resolution shell? Thank you. Best, Melody -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: [EMAIL PROTECTED] Phone: + 44 208 816 2515
Re: [ccp4bb] question about processing data
well, redundancy for the highest shell is 4.8, I/sigma is 3, Rmerge for overall is 0.08 for highest shell is 0.336. I/sigma and Rmerge don't seem quite nice... thanks. On Mon, Mar 17, 2008 at 11:51 AM, Partha Chakrabarti [EMAIL PROTECTED] wrote: Hi Melody, There was a nice discussion in this year's ccp4 study weekend. In general, one needs to consider several factors.. If you were at 3A, or low symmetry, you would of course try to get the maximum out of it, on the other hand, there are requirements for experimental phasing.. in general, judge it from: 1. Completeness 2. Redundancy 3. I / Sigma 4. R merge statistics Not just one of them. If you are pushing it too far, you will see the effect in later refinement step.. With 74% completeness, how does the other parameters look like? HTH, Partha On Mon, Mar 17, 2008 at 10:06 AM, Melody Lin [EMAIL PROTECTED] wrote: Hi all, I have always been wondering... for a data set diffracting to say 2.15 Angstrom but in the highest resolution shell (2.25-2.15) the completeness is 74%, should I use merge all the data and call it a 2.15 A dataset or I should cut the data set to say 2.25 A where the highest resolution shell has better completeness (85%)? What is an acceptable completeness value for the highest resolution shell? Thank you. Best, Melody -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: [EMAIL PROTECTED] Phone: + 44 208 816 2515
Re: [ccp4bb] question about processing data
Looks ok I guess.. for the highest shell, if Rmerge is less than 0.45 and I/sigma is about 2, it is worth a try.. as James said, completeness might be from why it is incomplete.. is it something like C2? experts might tell us more.. Best, Partha On Mon, Mar 17, 2008 at 11:03 AM, Melody Lin [EMAIL PROTECTED] wrote: well, redundancy for the highest shell is 4.8, I/sigma is 3, Rmerge for overall is 0.08 for highest shell is 0.336. I/sigma and Rmerge don't seem quite nice... thanks. On Mon, Mar 17, 2008 at 11:51 AM, Partha Chakrabarti [EMAIL PROTECTED] wrote: Hi Melody, There was a nice discussion in this year's ccp4 study weekend. In general, one needs to consider several factors.. If you were at 3A, or low symmetry, you would of course try to get the maximum out of it, on the other hand, there are requirements for experimental phasing.. in general, judge it from: 1. Completeness 2. Redundancy 3. I / Sigma 4. R merge statistics Not just one of them. If you are pushing it too far, you will see the effect in later refinement step.. With 74% completeness, how does the other parameters look like? HTH, Partha On Mon, Mar 17, 2008 at 10:06 AM, Melody Lin [EMAIL PROTECTED] wrote: Hi all, I have always been wondering... for a data set diffracting to say 2.15 Angstrom but in the highest resolution shell (2.25-2.15) the completeness is 74%, should I use merge all the data and call it a 2.15 A dataset or I should cut the data set to say 2.25 A where the highest resolution shell has better completeness (85%)? What is an acceptable completeness value for the highest resolution shell? Thank you. Best, Melody
Re: [ccp4bb] Missing reflections
Can anyone explain the rationale for treating the test set reflections as 'unobserved' for the maps, even though they have perfectly good observed Fo values? This doesn't make a great deal of sense to me! Looking at the mtzdump output for the MTZ file output by Refmac, I indeed note that for these reflections, the amplitude for the weighted difference Fourier (i.e. column labelled DELFWT) is set to zero, and the amplitude for the weighted Fourier (i.e. column labelled FWT) is set to D.Fc (which is at least logical since if we set Fo such that mFo-DFc = 0 then mFo = DFc and so 2mFo-DFc = DFc). But it would seem more sensible to compute the coefficients based on the observed Fo since we have them! My rationale for this would be: in as much as the maps obtained from a refinement with the test set excluded do not reflect the 'final' density for publication, which I think is generally agreed should be obtained from a final refinement using all data (working + test set), the maps using the observed mFo instead of DFc for the test set would reflect the true differences between this and the final maps, i.e. they would indicate the true effect of omitting the test set. I accept there is a practical problem here in that the sigmaA values which are needed to compute the map coefficients are only unbiased if computed from the test set, but a way around this would be to carry forward the sigmaA values from the penultimate refinement excluding the test set and use those in the final refinement using all data. This is not a perfect solution, but it's better than nothing. -- Ian -Original Message- From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of George M. Sheldrick Sent: 12 March 2008 16:01 To: Simon Kolstoe Cc: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Missing reflections All these programs only refine against reflections that were actually measured. REFMAC, but not SHELXL, provides the 'Sigma-A' weight coefficients for Coot to use DFc instead of 2mFo-DFc for the reflections for which Fo is not known (or is reserved for the free R) to calculate a map. This will in general improve the appearence of the map at the cost of introducing a little model bias. As far as I know these 'unobserved' reflections are not used in calculating the difference map. CNS is probably like SHELXL, I'm not sure what phenix.refine does. George Prof. George M. Sheldrick FRS Dept. Structural Chemistry, University of Goettingen, Tammannstr. 4, D37077 Goettingen, Germany Tel. +49-551-39-3021 or -3068 Fax. +49-551-39-2582 On Wed, 12 Mar 2008, Simon Kolstoe wrote: Dear CCP4bb, I was looking through the REFMAC manual today and found the following advice: Completing the data to include all possible hkls. Should do this after data reduction, and certainly before using REFMAC. This is now done with the uniqueify script. It is best done using CCP4i. http://www.ccp4.ac.uk/dist/html/refmac5/usage/examples.html#exam0 Is it a good idea to always run uniqueify on data before running REFMAC - what about other refinement programs such as SHELX, CNS or phenix.refine? Simon Disclaimer This communication is confidential and may contain privileged information intended solely for the named addressee(s). It may not be used or disclosed except for the purpose for which it has been sent. If you are not the intended recipient you must not review, use, disclose, copy, distribute or take any action in reliance upon it. If you have received this communication in error, please notify Astex Therapeutics Ltd by emailing [EMAIL PROTECTED] and destroy all copies of the message and any attached documents. Astex Therapeutics Ltd monitors, controls and protects all its messaging traffic in compliance with its corporate email policy. The Company accepts no liability or responsibility for any onward transmission or use of emails and attachments having left the Astex Therapeutics domain. Unless expressly stated, opinions in this message are those of the individual sender and not of Astex Therapeutics Ltd. The recipient should check this email and any attachments for the presence of computer viruses. Astex Therapeutics Ltd accepts no liability for damage caused by any virus transmitted by this email. E-mail is susceptible to data corruption, interception, unauthorized amendment, and tampering, Astex Therapeutics Ltd only send and receive e-mails on the basis that the Company is not liable for any such alteration or any consequences thereof. Astex Therapeutics Ltd., Registered in England at 436 Cambridge Science Park, Cambridge CB4 0QA under number 3751674
[ccp4bb] 2008 Gordon Research Conference on Diffraction Methods in Structural Biology
Gordon Research Conference on Diffraction Methods in Structural Biology July 13-18, 2008, Bates College, Lewiston, Maine, USA Co-Chairs: Elspeth Garman Andrew Leslie The 2008 Gordon Research Conference on Diffraction Methods in Structural Biology will encompass advances in the methodology for macromolecular X-ray crystallography, and other diffraction/scattering applications. The full confirmed programme and timetable for the meeting can now be found at: http://www.grc.org/programs.aspx?year=2008program=diffrac as well as details on how to register (registration closes on 22nd June 2008 but attendance is limited to 125 researchers). All macromolecular crystallographers interested in Methods development are encouraged to consider taking part in this meeting. Participants are expected to contribute to discussion, to present a poster, and to attend the entire conference. - Dr. Elspeth F. Garman, Reader in Molecular Biophysics, University of Oxford Visiting Professor in Chemistry, University of Durham Postal address: Department of Biochemistry, Rex Richards Building, University of Oxford, Tel: (44)-1865-275398 South Parks Road, FAX: (44)-1865-275182 OXFORD, OX1 3QU, U.K. E-mail: [EMAIL PROTECTED] -
[ccp4bb] problem installing arp/warp
Dear all, I am trying to install arp/warp and I am stuck with the following error: -- Checking refmac5 installation - refmac5: Command not found. *** ERROR *** Cannot execute refmac5 *** INSTALLATION OF ARP/wARP 7.0.1 FAILED *** -- CCP4 is installed and refmac5 is running. A few more information: The system is Kubuntu 7.10 My shell is bash (tried tcsh as well and got the same error) Typing refmac5 -i returns: -- CCP4 software suite: library version 6.0 CCP4 software suite: patch level 6.0.2 Program: refmac5; version 5.2.0019 -- when I type refmac5 I get: -- ### ### ### ### CCP4 6.0: Refmac_5.2.0019version 5.2.0019 : 06/09/05## ### User: manager Run date: 17/ 3/2008 Run time: 09:45:33 Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. !--SUMMARY_END--/FONT/B $TEXT:Reference1: $$ comment $$ Refinement of Macromolecular Structures by the Maximum-Likelihood Method: G.N. Murshudov, A.A.Vagin and E.J.Dodson,(1997) Acta Crystallogr. D53, 240-255 EU Validation contract: BIO2CT-92-0524 $$ $SUMMARY :Reference1: $$ Refmac: $$ :TEXT:Reference1: $$ -- Thank you all in advance for any help.
Re: [ccp4bb] exclude range within data in scala , discontinuous run in scala problem
Hi Phil,Thanks for your email. I did get it to work with the options you
suggested .
i.e
Toggle ON Override automatic definition of runs to mark discontinuities in
data
Toggle ON Define Runs
Toggle OFF Use run 1 as reference run.
Setup runs as follows ( intended to exclude batches 200 to 400 from a total
dataset of 1 to 720 )
Run 1 from 1 to 200
Run 2 from 400 to 720
This combination worked as you said it would.
Thank you for your help
Hari Jayaram
Postdoc
Brandeis University
On Sat, Mar 15, 2008 at 3:44 AM, Phil Evans [EMAIL PROTECTED] wrote:
You are right: this is a bug I should fix sometime
Assigning two runs should work though
You should not assign a reference run, and you shouldn't need to
reassign the datasets
Best wishes
Phil
On 14 Mar 2008, at 21:45, hari jayaram wrote:
Hi.
I did try that beforehand when I tried excluding a range of
batches with the ccp4i gui
But I got an error
Scala: *** Gap in time (rotation) ***
Sorry...both versions of the protocol for handling a bad internal
wedge are giving me either a gap in rotation error or a Run 2
has not been assigned to a dataset error
I am still stuck.
( error and com file for the exclude data range option is attached
below)
Thanks for your help.
Hari Jayaram
Error
---
Large gap in time (rotation) coordinate:3.5
See WARNING above
Smoothed B-factor impossible
***
* Information from CCP4Interface script
***
The program run with command: scala HKLIN /Users/hari/aps_feb08/
p2-2/p2-2_A1_1_0001_sorted.mtz HKLOUT /tmp/hari/p2_2_13_2_mtz.tmp
SCALES /Users/hari/aps_feb08/p2-2/p2_2_13.scala ROGUES /Users/
hari/aps_feb08/p2-2/p2_2_13_rogues.log NORMPLOT /Users/hari/
aps_feb08/p2-2/p2_2_13_normplot.xmgr ANOMPLOT /Users/hari/
aps_feb08/p2-2/p2_2_13_anomplot.xmgr PLOT /Users/hari/aps_feb08/
p2-2/p2_2_13_surface_plot.plt CORRELPLOT /Users/hari/aps_feb08/
p2-2/p2_2_13_correlplot.xmgr
has failed with error message
Scala: *** Gap in time (rotation) ***
***
#CCP4I TERMINATION STATUS 0 Scala: *** Gap in time (rotation)
***
#CCP4I TERMINATION TIME 14 Mar 2008 17:38:34
#CCP4I TERMINATION OUTPUT_FILES /Users/hari/aps_feb08/p2-2/
p2-2_A1_1_0001_sorted.mtz p2_2
#CCP4I MESSAGE Task failed
The com script was
***
/tmp/hari/p2_2_13_3_com.tmp
***
title Scala anon and deleted batches 200_400b try with two run
definitions
name project p2_2 crystal p2-2_A1_1 dataset p2_2
exclude EMAX -
10.0
exclude batch -
400 to 540
partials -
check -
test 0.95 1.05 -
nogap
intensities PROFILE -
PARTIALS
final PARTIALS
scales -
rotation SPACING 5 -
secondary 6 -
bfactor ON -
BROTATION SPACING 20
UNFIX V
FIX A0
UNFIX A1
initial MEAN
tie surface 0.001
tie bfactor 0.3
cycles 10 converge 0.3 reject 2
anomalous on
output AVERAGE
print cycles nooverlap
RSIZE 80
## This script run with the command ##
# scala HKLIN /Users/hari/aps_feb08/p2-2/p2-2_A1_1_0001_sorted.mtz
HKLOUT /tmp/hari/p2_2_13_2_mtz.tmp SCALES /Users/hari/aps_feb08/
p2-2/p2_2_13.scala ROGUES /Users/hari/aps_feb08/p2-2/
p2_2_13_rogues.log NORMPLOT /Users/hari/aps_feb08/p2-2/
p2_2_13_normplot.xmgr ANOMPLOT /Users/hari/aps_feb08/p2-2/
p2_2_13_anomplot.xmgr PLOT /Users/hari/aps_feb08/p2-2/
p2_2_13_surface_plot.plt CORRELPLOT /Users/hari/aps_feb08/p2-2/
p2_2_13_correlplot.xmgr
On Fri, Mar 14, 2008 at 5:11 PM, Phil Evans [EMAIL PROTECTED]
wrote:
In ccp4i Scala task, click to open the Excluded data panel, click on
Exclude selected batches
There you can define one or more ranges of batches or lists to exclude
If you just want to exclude the last part you can define a range eg
301 to 999
You don't need to explicitly define runs
Phil
On 14 Mar 2008, at 18:57, hari jayaram wrote:
Hi I am trying to exclude a bad wedge of data during scaling in
scala in the newest ccp4
( fink install this morning from W.G Scotts sage.ucsc Binaries ..so
they should be version 6)
The batches I need are
1 to 200 and 400-720
I have clicked the Override automatic definition of runs to mark
discontinuities in data button as well as created two runs to
contain the required data
But I get a Run 2 has not been assigned to a dataset error.
How I can exclude a bad wedge
Re: [ccp4bb] question about processing data
Melody Lin wrote: Hi all, I have always been wondering... for a data set diffracting to say 2.15 Angstrom but in the highest resolution shell (2.25-2.15) the completeness is 74%, should I use merge all the data and call it a 2.15 A dataset or I should cut the data set to say 2.25 A where the highest resolution shell has better completeness (85%)? What is an acceptable completeness value for the highest resolution shell? Thank you. Best, Melody Hi Melody, This reply is not aimed at you directly as this situation seems to have become systemic in the field. So thanks for bringing it up! We can have a long, and mostly aimless, discussion on what resolution you should claim for your data set but DON'T throw away good data to make the statistics look better. At high resolution the statistics are supposed to get worse! What matters is if the data still contain useful information. The fact that 26% of the data is missing does not normally mean that anything is wrong with the 74% that you did measure. Perhaps you used a square detector and didn't place it close enough to capture the full resolution, or perhaps your diffraction pattern is anisotropic. The only reason to throw out data is if they are too inaccurate for your purpose. When your data is used for phasing, especially anomalous phasing, there is reason to focus on data quality, but I see far too many native data sets that make poor use of the diffraction potential of the crystal. I thought this was due to people not properly collecting the data, but now it seems that people are simply throwing away good data because they don't like the statistics. So my advice; if your high resolution shell data has poor completeness then check why this happened. If you did not collect the data properly then let it be a lesson for the next data collection trip. If it resulted from some issue of the crystal then decide if the measured data is messed up as well. If not then use all the data you trust, which means there is useful signal (I/SigI 1.5 or 2.0 depending who you talk to) and no problems leading to systematic errors or outliers. Bart == Bart Hazes (Assistant Professor) Dept. of Medical Microbiology Immunology University of Alberta 1-15 Medical Sciences Building Edmonton, Alberta Canada, T6G 2H7 phone: 1-780-492-0042 fax:1-780-492-7521 ==
Re: [ccp4bb] question about processing data
On Mon, 2008-03-17 at 10:51 +, Partha Chakrabarti wrote: Not just one of them. If you are pushing it too far, you will see the effect in later refinement step.. And the effect in later refinement step will be the slight increase in R-factor? IMHO, this does not justify throwing away data (which ultimately reduces the quality of your model). -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] question about processing data
I would use all the data myself and report that the model was built from a a dataset with 74% completeness in the 2.25 to 2.15 Anngstrom shell. I would not put the number 2.15 A in the manuscript title nor in the poster title. For me the acceptable completeness is 90% in the highest resolution shell for the number to get in the title. You will know I reviewed your paper if you see my telltale reviewer comment. You can put whatever you want in the PDB deposition field. Jim On Mon, 17 Mar 2008, Melody Lin wrote: Hi all, I have always been wondering... for a data set diffracting to say 2.15Angstrom but in the highest resolution shell ( 2.25-2.15) the completeness is 74%, should I use merge all the data and call it a 2.15 A dataset or I should cut the data set to say 2.25 A where the highest resolution shell has better completeness (85%)? What is an acceptable completeness value for the highest resolution shell? Thank you. Best, Melody
Re: [ccp4bb] problem installing arp/warp
Dear Mario, it seems that inside the install script the path variable is not set and therefore the call to refmac5 fails. We are not sure why this happend in your case. The install script was tested with both (t)csh and bash and something in your machine setup was not anticipated by us. Have you run the install.sh utility in the same terminal/shell, in which you later verified your refmac installation by typing 'refmac5 -i'? Could it be that you need to run some setup util for ccp4 manually before? To get you started with ARP/wARP we can send you a taylored install script with some diagnostic output to find a fix. In the meanwhile, can you try to change the line alias runtestrefmac 'refmac5 -i' (line 16 in install_csh.sh) to incorporate the absolute path to the refmac5 binary? Which refmac5 should provide you with this. Regards, Gerrit and Victor. Mario Sanches wrote: Dear all, I am trying to install arp/warp and I am stuck with the following error: -- Checking refmac5 installation - refmac5: Command not found. *** ERROR *** Cannot execute refmac5 *** INSTALLATION OF ARP/wARP 7.0.1 FAILED *** -- CCP4 is installed and refmac5 is running. A few more information: The system is Kubuntu 7.10 My shell is bash (tried tcsh as well and got the same error) Typing refmac5 -i returns: -- CCP4 software suite: library version 6.0 CCP4 software suite: patch level 6.0.2 Program: refmac5; version 5.2.0019 -- when I type refmac5 I get: -- ### ### ### ### CCP4 6.0: Refmac_5.2.0019version 5.2.0019 : 06/09/05## ### User: manager Run date: 17/ 3/2008 Run time: 09:45:33 Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. !--SUMMARY_END--/FONT/B $TEXT:Reference1: $$ comment $$ Refinement of Macromolecular Structures by the Maximum-Likelihood Method: G.N. Murshudov, A.A.Vagin and E.J.Dodson,(1997) Acta Crystallogr. D53, 240-255 EU Validation contract: BIO2CT-92-0524 $$ $SUMMARY :Reference1: $$ Refmac: $$ :TEXT:Reference1: $$ -- Thank you all in advance for any help.
[ccp4bb] Post-doctoral job opportunity
All, Below you will find the pertinent information for a job opening at the Howard Hughes Medical Research Institute. All information can be found at this site: http://www.hhmi.org/jobs/main?action=jobjob_id=548. If you are interested, please follow the instructions in the advertizement and do NOT e-mail applications to me. Thanks, Mark Mark van der Woerd, PhD Research Scientist II Dept. of Biochemistry and Molecular Biology Colorado State University Fort Collins, CO 80523 Phone (970) 491-0469 ? Job Summary: Looking for a highly motivated individual with a strong interest in integrated approaches to problems in structural biology. The lab has extensive crystallographic and spectroscopic resources, and is part of the W.M. Keck Center for Chromatin Structure and Function. Principal Responsibilities: Investigate the structure, function, and dynamic properties of eukaryotic chromatin, using a wide variety of biochemical, biophysical and in vivo approaches. Investigate how cellular or viral factors interact with histones, nucleosomes, or chromatin and how these interactions may lead to cancer or other diseases. Use multipronged approaches such as x-ray crystallography, small angle x-ray scattering, atomic force microscopy, fluorescence spectroscopy, analytical untracentrifugation, and methods in mechanistic biochemistry/molecular biology as well as genetic approaches to understand the mechanism by which structural transitions in chromatin occur. Preferred Qualifications: Ph.D. in Molecular Biology, Biochemistry, Biophysics or an appropriately related field. Researchers with a strong background in biochemistry preferred. Previous experience with the structure determination of protein/protein and/or nucleic-acid complexes preferred. Extensive biochemical experience with protein purification, functional characterization, and yeast genetics would be highly valued.? Applicants should be strongly motivated, ambitious and function well in a highly collaborative environment. Additional Information: Please send cover letter, CV, and names of three references to Dr. Karolin Luger. Be sure to reference job #099100-01. To Apply To apply for this position, please email or send your resume to: Dr. Karolin Luger, PhD Investigator HHMI at Colorado State University Dept of Biochemistry Molecular Biology 1870 Campus Delivery, 383 MRB Fort Collins, Colorado 80523-1870 E-mail: [EMAIL PROTECTED] Application Deadline: Open Until Filled We are an Equal Opportunity Employer
[ccp4bb] Crystallization and Biophysics Courses
Dear all, We would like to announce that we are organizing two courses on: Biophysical Characterisation of Macromolecules (20-23 May) HTP Crystallization and Information Management (18-20 June) Both will take place at the NKI at Amsterdam, at our brand new lab space (we have not even moved yet ...)! More information for the respective course goals and preliminary schedules can be found at http://xtal.nki.nl ... and for even more events and conferences do not forget to look at: http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/ Current_events Tassos
Re: [ccp4bb] problem installing arp/warp
Dear Gerrit, Changing the alias to include the full path for refmac5 worked. Thank you very much for your help. I don't know if it helps you somehow, but I was running it on the same terminal where I typed refmac5 -i. Actually, the paths for the CCP4 programs are configured in /etc/bash.bashrc so that they will be visible everywhere for all users, so I think that was not the issue here. Thank you again for the quick reply, Mario Sanches Gerrit Langer wrote: Dear Mario, it seems that inside the install script the path variable is not set and therefore the call to refmac5 fails. We are not sure why this happend in your case. The install script was tested with both (t)csh and bash and something in your machine setup was not anticipated by us. Have you run the install.sh utility in the same terminal/shell, in which you later verified your refmac installation by typing 'refmac5 -i'? Could it be that you need to run some setup util for ccp4 manually before? To get you started with ARP/wARP we can send you a taylored install script with some diagnostic output to find a fix. In the meanwhile, can you try to change the line alias runtestrefmac 'refmac5 -i' (line 16 in install_csh.sh) to incorporate the absolute path to the refmac5 binary? Which refmac5 should provide you with this. Regards, Gerrit and Victor. Mario Sanches wrote: Dear all, I am trying to install arp/warp and I am stuck with the following error: -- Checking refmac5 installation - refmac5: Command not found. *** ERROR *** Cannot execute refmac5 *** INSTALLATION OF ARP/wARP 7.0.1 FAILED *** -- CCP4 is installed and refmac5 is running. A few more information: The system is Kubuntu 7.10 My shell is bash (tried tcsh as well and got the same error) Typing refmac5 -i returns: -- CCP4 software suite: library version 6.0 CCP4 software suite: patch level 6.0.2 Program: refmac5; version 5.2.0019 -- when I type refmac5 I get: -- ### ### ### ### CCP4 6.0: Refmac_5.2.0019version 5.2.0019 : 06/09/05## ### User: manager Run date: 17/ 3/2008 Run time: 09:45:33 Please reference: Collaborative Computational Project, Number 4. 1994. The CCP4 Suite: Programs for Protein Crystallography. Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. !--SUMMARY_END--/FONT/B $TEXT:Reference1: $$ comment $$ Refinement of Macromolecular Structures by the Maximum-Likelihood Method: G.N. Murshudov, A.A.Vagin and E.J.Dodson,(1997) Acta Crystallogr. D53, 240-255 EU Validation contract: BIO2CT-92-0524 $$ $SUMMARY :Reference1: $$ Refmac: $$ :TEXT:Reference1: $$ -- Thank you all in advance for any help.
Re: [ccp4bb] question about processing data
Redundancy of 4.8 for a 74% complete shell (if I understand which shell these stats are for) suggests you have assumed too much symmetry and are rejecting a lot of reflections during scaling. Is this the case? The I/sigma suggests you could drop the symmetry and re-scale without losing a lot of data if this is the case. On Mar 17, 2008, at 4:03 AM, Melody Lin wrote: well, redundancy for the highest shell is 4.8, I/sigma is 3, Rmerge for overall is 0.08 for highest shell is 0.336. I/sigma and Rmerge don't seem quite nice... thanks. On Mon, Mar 17, 2008 at 11:51 AM, Partha Chakrabarti [EMAIL PROTECTED] wrote: Hi Melody, There was a nice discussion in this year's ccp4 study weekend. In general, one needs to consider several factors.. If you were at 3A, or low symmetry, you would of course try to get the maximum out of it, on the other hand, there are requirements for experimental phasing.. in general, judge it from: 1. Completeness 2. Redundancy 3. I / Sigma 4. R merge statistics Not just one of them. If you are pushing it too far, you will see the effect in later refinement step.. With 74% completeness, how does the other parameters look like? HTH, Partha On Mon, Mar 17, 2008 at 10:06 AM, Melody Lin [EMAIL PROTECTED] wrote: Hi all, I have always been wondering... for a data set diffracting to say 2.15 Angstrom but in the highest resolution shell (2.25-2.15) the completeness is 74%, should I use merge all the data and call it a 2.15 A dataset or I should cut the data set to say 2.25 A where the highest resolution shell has better completeness (85%)? What is an acceptable completeness value for the highest resolution shell? Thank you. Best, Melody -- MRC National Institute for Medical Research Division of Molecular Structure The Ridgeway, NW7 1AA, UK Email: [EMAIL PROTECTED] Phone: + 44 208 816 2515 -- James Stroud UCLA-DOE Institute for Genomics and Proteomics Box 951570 Los Angeles, CA 90095 http://www.jamesstroud.com
Re: [ccp4bb] question about processing data
Hi - I would tend to argue as follows: An I/sigI of 3, and Rmerge of 33.6% are most definitely acceptable values with a redundancy of 4.8. Thus, despite the 74% completeness, that data are most definitely useful and should be included in refinement. A good question now is why is the data only 74% complete. I can think of a few reasons, eg 1. not enough 'degrees' collected in total: too bad, better do better next time, but thats not likely to be your problem. 2. overlaps at high resolution: again be more careful next time, but could you play with the mosaicity to decrease overlaps a bit ? 3. High resolution collected in the corners of detector: put the detector closer next time and dont collect data at the corners ... 4. Severe anisotropy: tough luck, have to live with it .. or try and deal better with it during data collection (adjust exposure) Whatever the case, I would use the data and clearly report in the MM in my paper not only what the numbers are, but also WHY they are like that. And, of course if its trivial to do a better data collection experiment and get the best data, as it often is, then do a better data collection experiment ... My main point is that you should know clearly WHY your high resolution shell is incomplete and then decide. The numbers alone do not always tell the full story. Best , Tassos well, redundancy for the highest shell is 4.8, I/sigma is 3, Rmerge for overall is 0.08 for highest shell is 0.336. I/sigma and Rmerge don't seem quite nice...
[ccp4bb] Coot (OS X) unable to read CRYST1 line
Hello all, I apologize for the off-topic post. I have a problem with Coot being unable to read the space group from the CRYST1 line in the PDB file. Although the space group is specified correctly, Coot seems unable to read it. It reads the unit cell dimensions and angles just fine - it seems to have a problem with just the space group. I use coot 0.4-pre-2 running on OS X Leopard on a Powerbook G4. The same PDB opens just fine on a Coot 0.4-pre-2 running on Redhat Linux Enterprise 5. The terminal window shows the following message when the pdb is opened: PDB file ABCD.pdb has been read. No Spacegroup found for this PDB file Cell: 55.76 118.43 122.38 107.8 98.36 91.4 !! Warning:: No symmetry available for this molecule No Symmetry for this model Molecule 0 read successfully Has anybody else come across this problem? Thanks! Pavan
Re: [ccp4bb] question about processing data
Dear all, Thank you very much for the useful suggestions! I definitely learned a lot from these discussions. Now looking back at my datasets, I think the incompleteness likely results from high mosaicity (1.009) and anisotropy of the crystal. Detector is square, but the distance is short enough for the resolution, and cell dimensions are not huge (~45x 90x 100 A), so there are not too much overlapping among high resolution spots. I am confident with the symmetry. Well, now I know better how to collect good data. Thanks! Best, Melody On Mon, Mar 17, 2008 at 8:29 PM, Anastassis Perrakis [EMAIL PROTECTED] wrote: Hi - I would tend to argue as follows: An I/sigI of 3, and Rmerge of 33.6% are most definitely acceptable values with a redundancy of 4.8. Thus, despite the 74% completeness, that data are most definitely useful and should be included in refinement. A good question now is why is the data only 74% complete. I can think of a few reasons, eg 1. not enough 'degrees' collected in total: too bad, better do better next time, but thats not likely to be your problem. 2. overlaps at high resolution: again be more careful next time, but could you play with the mosaicity to decrease overlaps a bit ? 3. High resolution collected in the corners of detector: put the detector closer next time and dont collect data at the corners ... 4. Severe anisotropy: tough luck, have to live with it .. or try and deal better with it during data collection (adjust exposure) Whatever the case, I would use the data and clearly report in the MM in my paper not only what the numbers are, but also WHY they are like that. And, of course if its trivial to do a better data collection experiment and get the best data, as it often is, then do a better data collection experiment ... My main point is that you should know clearly WHY your high resolution shell is incomplete and then decide. The numbers alone do not always tell the full story. Best , Tassos well, redundancy for the highest shell is 4.8, I/sigma is 3, Rmerge for overall is 0.08 for highest shell is 0.336. I/sigma and Rmerge don't seem quite nice...
Re: [ccp4bb] Coot (OS X) unable to read CRYST1 line
your CRYST1 card is most likely missing the actual space group name. You can fix this with e.g. pdbset: pdbset xyzin your.pdb xyzout pdb-with-spacegroup-name.pdb eof spac P21212 end eof where you replace P21212 with your actual space group name. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Mon, 17 Mar 2008, Pavan wrote: Hello all, I apologize for the off-topic post. I have a problem with Coot being unable to read the space group from the CRYST1 line in the PDB file. Although the space group is specified correctly, Coot seems unable to read it. It reads the unit cell dimensions and angles just fine - it seems to have a problem with just the space group. I use coot 0.4-pre-2 running on OS X Leopard on a Powerbook G4. The same PDB opens just fine on a Coot 0.4-pre-2 running on Redhat Linux Enterprise 5. The terminal window shows the following message when the pdb is opened: PDB file ABCD.pdb has been read. No Spacegroup found for this PDB file Cell: 55.76 118.43 122.38 107.8 98.36 91.4 !! Warning:: No symmetry available for this molecule No Symmetry for this model Molecule 0 read successfully Has anybody else come across this problem? Thanks! Pavan
Re: [ccp4bb] Summary: Calculating R-factor and maps from a Refmac model containing TLS downloaded from the PDB
Hi again, I guess this is only a partial summary, since I still don't understand all the issues this question raises. Pavel Afonine reported that his extensive tests of the PDB reveals that reproducing R values from models with TLS ADP's is a wide-spread and serious problem. The principal problems (IMHO) are 1) Incorrect or illegal TLS definitions in the REMARK. 2) Some files list in the ATOM B column the residual B after TLS has been accounted for while others list the total B (TLS and residual). There is no clear indication in the PDB file which interpretation is being used. Tassos, Eleanor, and others recommended taking the TLS definition from the PDB header and running zero cycles of unrestrained refinement in Refmac to get it to calculate R factors and Maps w/o the need to define ideal geometry for co-factors. I have yet to see this work, however (See below) Ulrich Baumann wrote to tell me of two of his PDB's that he knows will give back the reported R values. They are 2qua and 2qub. I grabbed 2qua from the RCSB server, extracted the TLS groups with CCP4i, and found that the TLS definitions were incorrect. There is one polypeptide in this model and three TLS groups. The first and third group did not have a residue range, while the second group defined a residue range in the middle of the peptide. I made the assumption that the first and third TLS groups were intended to cover the beginning and end of the peptide and corrected the .tls file. I loaded this into Refmac and asked for zero cycles of unrestrained refinement and got an R value of 19.4%. The PDB file says it should be 17.3%. I then asked Refmac to run 10 cycles of TLS and 10 cycles of restrained refinement and got an R value of 17.5%. Good enough. From this result I infer that Refmac is unable to calculate the original ADP's given this PDB file and TLS definition. It can reconstruct them via refinement, basically ignoring the B values of the PDB file. This particular PDB entry appears to contain in its B column the residual B's. I also tried entry 2qub, but with less luck. This model has seven peptides and 30 TLS groups. The first seven TLS groups defined in the header of the PDB cover each of the seven chains, while the other 23 groups had no residue range. I can guess that the intension was to have five TLS groups for each of the seven chains, but without additional information from Dr. Baumann, I'm unable to even start trying to reproduce R values and calculate maps. So... 1) Pavel is correct, there are many clear errors in the TLS REMARKs of PDB entries. 2) It seems necessary to ask Refmac to recreate the ADP description for a PDB entry from scratch, assuming the TLS group definition can be deduced from the PDB header. This, currently, requires refinement which requires .cif's for the unusual groups. If CCP4I could ask Refmac to perform only TLS/B refinement, holding positions fixed, the need for detailed .cifs would be greatly reduced. I have no desire to move the atoms anyway. Better yet, if someone could find out what Refmac is expecting to find in its starting PDB (what it wants in the B column) one could add a tool to CCP4I that could convert a PDB entry to what Refmac wants w/o refinement. Since there appear to be two varieties of entries one could try both possibilities and choose the one with the lowest R value. I have to close with additional problems, I'm afraid. I can't run the required refinement on 1nkz to test TLS/B refinement but I have tried it on 3bsd, where I have a good .cif for the Bchl-a groups. When I pull out the TLS definition, and perform 10 cycles of TLS and 10 cycles of restrained refinement I get an R value of 20.2% while the entry asserts that the correct value is 17.8%. The final TLS parameters look, by eye, pretty similar to the deposited ones, so I don't know what is going on here. Dale Tronrud Dale Tronrud wrote: Hi, I am looking over a number of models from the PDB but have been unable to reproduce the R-factors for any model that was refined with Refmac and contains TLS parameters. I usually can't get within 5% of the reported value. On the other hand, I usually do pretty well for models w/o TLS. An example is the model 1nkz. The PDB header gives an R value of 17% but even when I use tlsanal in CCP4i to generate a PDB with anisotropic B's that mimic the TLS parameters I get an R value of 22.4% using SFCheck. (I'm not implying that I suspect any problem with 1nkz, in fact I have every reason to believe this is the great model its published stats indicate.) I've found a CCP4 BB letter that stated that SFCheck does not pay attention to anisotropic B's but that letter was dated 2002. I hope this limitation has been removed, or at least the output would mention this limitation. Setting up a refinement in Refmac involves a large overhead, since even for zero cycles of refinement the program insists on a complete
Re: [ccp4bb] Summary: Calculating R-factor and maps from a Refmac model containing TLS downloaded from the PDB
On Monday 17 March 2008 16:20, Dale Tronrud wrote: Hi again, I guess this is only a partial summary, since I still don't understand all the issues this question raises. Pavel Afonine reported that his extensive tests of the PDB reveals that reproducing R values from models with TLS ADP's is a wide-spread and serious problem. The principal problems (IMHO) are 1) Incorrect or illegal TLS definitions in the REMARK. Yes. I have noticed the same. It is unfortunate, and not at all clear at what point the error creeps in. 2) Some files list in the ATOM B column the residual B after TLS has been accounted for while others list the total B (TLS and residual). There is no clear indication in the PDB file which interpretation is being used. That is a fundamental deficiency in the existing PDB standard. It simply doesn't specify how to present this critical information. A draft change covering this was circulated at the PDB get-together of last summer's ACA meeting, and I discussed with Garib and Eleanor that we should as a community decide how we would like it handled. The consensus as I understand it is that people would prefer that the B field of individual ATOM records contain the *net* B rather than the residual B, so that old programs will continue to work as expected. However, this puts even more importance on the TLS description in the header being correct, since the information is otherwise not recoverable. We were going to circulate a letter, but I plead guilty to letting the matter slide. Tassos, Eleanor, and others recommended taking the TLS definition from the PDB header and running zero cycles of unrestrained refinement in Refmac to get it to calculate R factors and Maps w/o the need to define ideal geometry for co-factors. I have yet to see this work, however (See below) Well, it has worked reasonably well for me in the past, for some structures. But it may well have broken again. Ulrich Baumann wrote to tell me of two of his PDB's that he knows will give back the reported R values. They are 2qua and 2qub. I grabbed 2qua from the RCSB server, extracted the TLS groups with CCP4i, and found that the TLS definitions were incorrect. There is one polypeptide in this model and three TLS groups. The first and third group did not have a residue range, while the second group defined a residue range in the middle of the peptide. I made the assumption that the first and third TLS groups were intended to cover the beginning and end of the peptide and corrected the .tls file. That is interesting, because the mmCIF file for that structure contains the following: # _pdbx_refine_tls_group.id 1 _pdbx_refine_tls_group.refine_tls_id 2 _pdbx_refine_tls_group.beg_auth_asym_idA _pdbx_refine_tls_group.beg_auth_seq_id 250 _pdbx_refine_tls_group.beg_label_asym_id A _pdbx_refine_tls_group.beg_label_seq_id252 _pdbx_refine_tls_group.end_auth_asym_idA _pdbx_refine_tls_group.end_auth_seq_id 461 _pdbx_refine_tls_group.end_label_asym_id A _pdbx_refine_tls_group.end_label_seq_id463 _pdbx_refine_tls_group.selection ? # This set of records is also a bit mangled, but does seem to contain additional traces of the correct residue ranges for each group. I wonder if the internal PDB database is storing incorrectly formatted XML descriptions of the groups, and then further corrupting the information when it generates a PDB format file? I also tried entry 2qub, but with less luck. Indeed. That one has no additional information in the mmCIF file either. So I don't know what's up. Here's a recent deposition of ours: 3BJE This one downloads from today's www.pdb.org with full TLS information. So the process clearly works at least some of the time. I have to close with additional problems, I'm afraid. I can't run the required refinement on 1nkz to test TLS/B refinement but I have tried it on 3bsd, where I have a good .cif for the Bchl-a groups. When I pull out the TLS definition, and perform 10 cycles of TLS and 10 cycles of restrained refinement I get an R value of 20.2% while the entry asserts that the correct value is 17.8%. The final TLS parameters look, by eye, pretty similar to the deposited ones, so I don't know what is going on here. The issue of proper TLS description is not the only difficulty in reproducing R factors from a PDB file. Another notable omission is the lack of scattering factors. If you have refined a SAS data set, e.g. a Se-edge dataset of a SeMet metallo-protein, then the R factors may vary by 1% just because of incorrectly reproduced f' terms for the Se and metal atoms. Ethan Merritt I loaded this into Refmac and asked for zero cycles of unrestrained refinement and got an R value of 19.4%. The PDB file says it should be 17.3%. I then asked Refmac to run 10 cycles of TLS and 10 cycles of restrained refinement and got an R value of 17.5%.
Re: [ccp4bb] Summary: Calculating R-factor and maps from a Refmac model containing TLS downloaded from the PDB
2) Some files list in the ATOM B column the residual B after TLS has been accounted for while others list the total B (TLS and residual). There is no clear indication in the PDB file which interpretation is being used. That is a fundamental deficiency in the existing PDB standard. It simply doesn't specify how to present this critical information. A draft change covering this was circulated at the PDB get-together of last summer's ACA meeting, and I discussed with Garib and Eleanor that we should as a community decide how we would like it handled. The consensus as I understand it is that people would prefer that the B field of individual ATOM records contain the *net* B rather than the residual B, so that old programs will continue to work as expected. However, this puts even more importance on the TLS description in the header being correct, since the information is otherwise not recoverable. We were going to circulate a letter, but I plead guilty to letting the matter slide. This is exactly what phenix.refine does (since 2005, I guess): it always prints out the total B-factor for each atom (Bindividual+Btls+Boverall). The TLS information (TLS matrices, origin coordinates and TLS group selections) are reported as well in PDB file header, so if necessary one can always extract the information about individual contributions. This makes it more straightforward to reproduce the R-factor statistics without any prior manipulations with the model. Another notable omission is the lack of scattering factors. If you have refined a SAS data set, e.g. a Se-edge dataset of a SeMet metallo-protein, then the R factors may vary by 1% just because of incorrectly reproduced f' terms for the Se and metal atoms. Ethan Merritt phenix.refine also always reports f' and f'' in output PDB file if they were used in refinement. I hope they don't get stripped off when deposited with PDB. Pavel.
[ccp4bb] Postdoctoral Fellowship at NIH Structural Biology of Neurotransmitter Receptors
A postdoctoral position is available to study the structure and function of neurotransmitter receptors. Recent papers describing work from the lab include: Nat Struct Mol Biol. 2006 13:1120-7; Nature. 2006 440 :456-462; Neuron 2007 53:829-841.We are looking for a highly motivated person who would like to combine crystallographic studies with biochemical and electrophysiological experiments to understand the function of glutamate receptor ion channels, the major mediators of excitatory synaptic transmission in the brain. A major component of the work involves protein expression and pre-crystallization screening. Strong skills in biochemistry and molecular biology are essential. Preference will be given to candidates with a proven track record using baculo virus, insect cell, and other eukaryotic expression systems. Must have excellent communication skills and a superb ability to work as part of a team. The research facilities at NIH are outstanding with regular access to the SER-CAT beamline at APS. Position available fall 2008. Initial duration 2 years, renewable for up to 5 years. J1 visa sponsorship for Non US Nationals To be eligible candidates must have a PhD awarded no more than 5 years ago. NIH is an EEO emplyer.
Re: [ccp4bb] Coot (OS X) unable to read CRYST1 line - Solved!
Once again, thanks a lot for all your replies. I just found out the problem. Ultimately it was quite a silly mistake - I had an old and defunct SYMINFO environment variable from solve/ resolve in my .cshrc file, which clashed with the one that coot was setting (in /sw/share/coot/setup/coot.sh). Once I removed the old SYMINFO reference, coot could open the PDB files properly. Thank you very much for all the help. Pavan On Mon, Mar 17, 2008 at 8:38 PM, Noinaj, Nicholas [EMAIL PROTECTED] wrote: just a quick question, have you just tried opening coot, then opening the *.pdb file from the file open menu? also, do you get errors when you just open coot alone, sometimes even single spaces where they shouldn't be makes a huge difference here. my next suggestion was (as you eluded to below) to open another file to see if any problems exist with it. hypothetically speaking, what happens if you simply remove the CRYST1 line? I am not as familiar with the OS version you are running, but with WinXP, i don't even need the CRYST1 card to view the structure, only when trying to view symmetry partners, etc. where it is needed. hope some of this helps...best of luck! I would be happy to look at your file here if you can shoot me the coordinates, or the first 50 lines or so, just enough to open in COOT. again, good luck! Cheers, Nick
