Re: [ccp4bb] regarding refinement and structure determination by MR
1. I would recommend to use Molprobity on the website: http://molprobity.biochem.duke.edu/ 2. Remember that if your spacegroup is P1, the origin (i.e. translations along a, b and c) is not determined and the first molecule may be placed anywhere. Similarly, in monoclinic spacegroups, the translation along b. is not determined. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 9 Jun 2011, at 09:46, Ting-Wei Jiang wrote: Dear experts, I got two questions regarding refinement and structure determination by MR. 1.I got a dataset of resolution at 2A refined to Rvalue~20% and Rfree~24% I want to know if there is any program in CCP4 could help me to check the refined structure in detail. For example,the model statistics in CNS could list some information that infer band,angle violation...etc Or I should ask what do I need to check before submitting to PDB. 2.A dataset of resolution collected to 2.2A is from native protein crystal(A) and its complex form(A'+B) whose PDB code is 2QN5 was already determined by MR(use another protein as search model).I planned to use A' as search model but the A' looks broken. This number of molecules in AU was predicted to 4~6 using Mathews coefficient. N/aM.C. solvent% 4 2.99 58.92 5 2.39 48.65 6 1.99 38.38 The coordinate or orientation of output pdb(as attached figure) from MR are always different since I changed parameter,such as different resolution,Multi copy,search mode...etc. even the contrast value of every run is pretty high(3) I really don't what happened with this dataset and any suggestion would be greatly appreciated. output-pdb.png
[ccp4bb] Diamond phase III proposal. Call for Expressions of Interest. Deadline looming!
Please don't hit delete yet, read on! First of all, many thanks to all the people who have already submitted a letter of support for the initiative Versatile microfocus beamline with a side-station for in-situ diffraction. It is essential that we get as many letters of support as possible, to show that the whole community feels that this is a much needed resource. So please send as many individual EOIs as possible, preferably not just one per group/institution. It is felt that it is also appropriate for PhD students and post-docs to express their interest in a beamline that will facilitate their future research. To make it easier for those who have no experience of writing such letters, some hints: Typically the text would cover: - That you're supporting this application - some words to show you know what it's about, including the title:Versatile microfocus beamline with a side-station for in-situ diffraction. - What is your area of science: can be quite specific, e.g. pathways of XYZ, chemical biology of ABC; but do say that your projects routinely end up at synchrotrons (Diamond). - What's difficult about it that the beamline will help resolve. Something like: obtaining diffraction quality crystals is routinely very difficult, so if diffraction quality were redefined, your life would be easier. To upload your letter of support, go to http://www.diamond.ac.uk/Home/Beamlines/PhaseIII/ExpressionsofInterest.html This link also provides more background to this and other proposals. This proposal is labelled 'P20'. The deadline is tomorrow, so do it now, it only takes 10 minutes. Cheers, Johan Dr. Johan P. Turkenburg X-ray facilities manager York Structural Biology Laboratory University of York Phone (+) 44 1904 328251 York YO10 5DD UK Fax (+) 44 1904 328266
Re: [ccp4bb] Two different asymmetric units from two different crystallization conditions
On 06/08/2011 07:19 PM, Shiva Bhowmik wrote: Dear All, I am working on a protein structure that yielded comparable diffraction quality crystals from two different crystallization condition. One of the crystallization condition conatins high conc. of salt pptant whereas the oher one contains high conc. of organic pptant. There are some minor differences in the stucture with respect to the backbone but what most surprising is the oligomeric structure. AnSEC study suggest the protein to be a tetramer in solution and a tetrameric assembly is observed in the asymmetric unit of the space group of the crystal obtained from high conc. of organic pptant. However, the crystal structure from the high conc. of salt pptant does not reveal any oligomeric assembly - symmetry operations of the space group does not suggest any oligomeric assembly. I believe the high salt conc. disrupted the tetrameric assembly and enabled crystallization of the protein as a monomer. Curious to know if there has been any similar precedence before. Cheers, Shiva I would submit both structures to PISA at the EBI, and look at the interfaces to see if there is any similarity between the 2 forms.. Eleanor
Re: [ccp4bb] regarding refinement and structure determination by MR
First Q. Checking the refined structure in detail.. This is personal. Basic - run REFMAC with monitor many - that lists really bad bonds, chirality, symmetry clashes etc, but frankly by the time you are at R=20% there shouldnt be many of those.. You need to be sure you have described any CIS peptides correctly - conflict can arise between REFMAC and COOT over whether something is or is not a CISPEP.. Then I use the COOT validation tools as a first and excellent start. Missing blobs, Difference map peaks Correct GLN/ASN etc. Look at ramachandran outliers - etc etc MOLPROBITY is great at the end - I think it results in overkill if you still have any gross errors to correct.. Q2. Kevin cowtan has written a wonderful utility called csymmatch. It moves a seciond solution to the same origin and symmetry operator as the first csymmatch -pdbin-ref oldone.pdb -pdbin newone.pdb -pdbout newone-shifted -origin-hand It is a great help after MR or automated model building.. You still have to match the chain IDs if you want A1 to match A2 etc, but it is a great help.. Eleanor On 06/09/2011 08:46 AM, Ting-Wei Jiang wrote: Dear experts, I got two questions regarding refinement and structure determination by MR. 1.I got a dataset of resolution at 2A refined to Rvalue~20% and Rfree~24% I want to know if there is any program in CCP4 could help me to check the refined structure in detail. For example,the model statistics in CNS could list some information that infer band,angle violation...etc Or I should ask what do I need to check before submitting to PDB. 2.A dataset of resolution collected to 2.2A is from native protein crystal(A) and its complex form(A'+B) whose PDB code is 2QN5 was already determined by MR(use another protein as search model).I planned to use A' as search model but the A' looks broken. This number of molecules in AU was predicted to 4~6 using Mathews coefficient. N/aM.C. solvent% 4 2.99 58.92 5 2.39 48.65 6 1.99 38.38 The coordinate or orientation of output pdb(as attached figure) from MR are always different since I changed parameter,such as different resolution,Multi copy,search mode...etc. even the contrast value of every run is pretty high(3) I really don't what happened with this dataset and any suggestion would be greatly appreciated.
Re: [ccp4bb] regarding refinement and structure determination by MR
Dear Ting-Wei, 1) Refmac writes model statistics like RMS bond lengths, angles etc. in the header of the ouput pdb. Open the output pdb in your favorite editor and look for REMARK 3 cards. There you find probably all the information you need. 2) You give very little details, so here I can only make some general remarks. I asume that your problem is that the electron density for A' looks broken? - check your diffraction images to make sure they are ok. Things to look for are ice rings, bad zones etc. I usually look at every 10th frame. - did you check for twinning? - do not asume the space group you got from your data processing package is correct. In molecular replacement you should test all space groups that are compatible with the cell dimensions. E.g. in phaser you can use the keyword SGALTERNATIVE ALL. - From the Mathews coefficients you give, I would say that 3 molecules in the AU are also possible, so you should look how the electron density looks with fewer molecules. - I guess that A' and B are both proteins? In that case your crystals could also contain only A' or only B molecules, so you should repeat the molecular replacement with only A' and with only B as search model. - It never hurts to collect another data set, preferably from a crystal grown under different conditions. Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ting-Wei Jiang Sent: Thursday, June 09, 2011 9:46 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] regarding refinement and structure determination by MR Dear experts, I got two questions regarding refinement and structure determination by MR. 1.I got a dataset of resolution at 2A refined to Rvalue~20% and Rfree~24% I want to know if there is any program in CCP4 could help me to check the refined structure in detail. For example,the model statistics in CNS could list some information that infer band,angle violation...etc Or I should ask what do I need to check before submitting to PDB. 2.A dataset of resolution collected to 2.2A is from native protein crystal(A) and its complex form(A'+B) whose PDB code is 2QN5 was already determined by MR(use another protein as search model).I planned to use A' as search model but the A' looks broken. This number of molecules in AU was predicted to 4~6 using Mathews coefficient. N/aM.C. solvent% 4 2.99 58.92 5 2.39 48.65 6 1.99 38.38 The coordinate or orientation of output pdb(as attached figure) from MR are always different since I changed parameter,such as different resolution,Multi copy,search mode...etc. even the contrast value of every run is pretty high(3) I really don't what happened with this dataset and any suggestion would be greatly appreciated.
Re: [ccp4bb] off-topic: Synchrotron look alike
Unfortunately it won't be in such a nice setting as ESRF though. http://www.linuxjournal.com/files/linuxjournal.com/linuxjournal/articles/036/3612/3612f1.jpg And who knows there might be an App for that (Remote Data Collection on your iPhone). I heard rumors that SSRL is implementing iRDC soon. Current features include display of actual SPEAR status and a timer when you should mount the next crystal. Jürgen On Jun 9, 2011, at 11:49 AM, Vellieux Frederic wrote: Sorry, a bit off topic. But in the news today (check google news for example): Steve Jobs (Apple) has revealed his plans for the new Apple campus (Apple headquarters) in Cupertino, California. Should look familiar to macromolecular crystallographers. Fred. apple.jpg .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/
Re: [ccp4bb] off-topic: Synchrotron look alike
Perhaps the shape is meant to amplify the Steve Jobs' Reality Distortion Field? On Thu, Jun 9, 2011 at 11:49 AM, Vellieux Frederic frederic.velli...@ibs.fr wrote: Sorry, a bit off topic. But in the news today (check google news for example): Steve Jobs (Apple) has revealed his plans for the new Apple campus (Apple headquarters) in Cupertino, California. Should look familiar to macromolecular crystallographers. Fred. -- Jim Fairman, Ph D. Post-Doctoral Fellow National Institutes of Health - NIDDK The Buchanan Lab http://www-mslmb.niddk.nih.gov/buchanan/index.html Lab: 1-301-594-9229 E-mail: fairman@gmail.com james.fair...@nih.gov
Re: [ccp4bb] off-topic: Synchrotron look alike
The sad thing is, although Macs are great crystallography platforms, stereo is hard at best, ridiculously expensive compared to Linux systems, and still requires the use of CRTs which have not been manufactured for years ...
Re: [ccp4bb] off-topic: Synchrotron look alike
On Thu, Jun 09, 2011 at 12:43:07PM -0400, Mayer, Mark (NIH/NICHD) [E] wrote: The sad thing is, although Macs are great crystallography platforms, stereo is hard at best, ridiculously expensive compared to Linux systems, and still requires the use of CRTs which have not been manufactured for years ... The Zalman passive stereo solution works nicely on Macs. That's our recommendation these days. -ben -- | Ben Eisenbraun | SBGrid Consortium | http://sbgrid.org | | Harvard Medical School | http://hms.harvard.edu |
Re: [ccp4bb] off-topic: Synchrotron look alike
Yeah, I know what you mean. That Zalman 3D LCD monitor put me back almost $300, and the mac mini I hooked it to, nearly another $600. My SGIs only cost $12K each in 1998. On Jun 9, 2011, at 9:43 AM, Mayer, Mark (NIH/NICHD) [E] wrote: The sad thing is, although Macs are great crystallography platforms, stereo is hard at best, ridiculously expensive compared to Linux systems, and still requires the use of CRTs which have not been manufactured for years ...
Re: [ccp4bb] off-topic: Synchrotron look alike
I still keep a bill of SGI Indigo memory extension from 64 Mb to 128Mb - $ 12,000 from Kingston in 1994 Friends, do not be niggards, life is much nicer today. Dr Felix Frolow Professor of Structural Biology and Biotechnology Department of Molecular Microbiology and Biotechnology Tel Aviv University 69978, Israel Acta Crystallographica F, co-editor e-mail: mbfro...@post.tau.ac.il Tel: ++972-3640-8723 Fax: ++972-3640-9407 Cellular: 0547 459 608 On Jun 9, 2011, at 19:52 , William Scott wrote: Yeah, I know what you mean. That Zalman 3D LCD monitor put me back almost $300, and the mac mini I hooked it to, nearly another $600. My SGIs only cost $12K each in 1998. On Jun 9, 2011, at 9:43 AM, Mayer, Mark (NIH/NICHD) [E] wrote: The sad thing is, although Macs are great crystallography platforms, stereo is hard at best, ridiculously expensive compared to Linux systems, and still requires the use of CRTs which have not been manufactured for years ...
Re: [ccp4bb] Fwd: Re: Question about the statistical analysis-might be a bit off topic
Kay, The Wikipedia's error propagation article in its Caveats and Warnings paragraph calls this a Cauchy distribution. And that sounds strange to me. p.d.f. of the Cauchy distribution is non-zero for any finite argument, whereas the p.d.f. for the inverse Gaussian must tend to zero at x-0, and is in fact this p(t)=exp(-(1-x0*t)**2/(2*s**2*t**2))/(t**2*s*sqrt(2*pi)) where t=1/x, and x0 and s are from original N(x0,s). This does NOT look like Cauchy distribution and I don't even see any limiting case whereby these two would match. What they have in common is that in both cases the variance is undefined. This is because when t-inf, the exponential term remains ~constant and the p.d.f.~1/t**2 (same as Cauchy). The mean can still be defined as finite by combining negative and positive domains in a careful manner, but truth is that both left- and right-side integrals are infinite. As for variance, p.d.f.*t**2-const at t-+-inf, which leads to essentially infinite variance, in accord with your numerical observations. Perhaps the lesson is to be aware that Wikipedia may contain errors. After all, I did win Tour de France and Stanley Cup in 1997 - just give me 10 minutes and then check the Wikipedia :) This is clearly an example where the first-order approximation breaks down, and common sense tells me that this happens because we may divide by numbers close to zero. But it still works with real experimental data (given that errors are small). If inverse value makes physical sense, then the direct value will not obey normal distribution as x=0 are prohibited. The approximation still works though if relative error is significantly less than 100%. Which would break down for weak reflectionds, but then inverse intensity really is not a meaningful thing. And it shows that it might be useful to think about error propagation, and not blindly apply the formulas. Exactly right. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs
