[ccp4bb] MR problem --molrep
Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja
Re: [ccp4bb] MR problem --molrep
Hi, I dare to say about the possible way to do molrep from my recent experience. You can choose rotation and translation function job at first and do the self rotation fuction (for multimer) after that.Each of these run will generate two different outputs *_rf.molrep_rf ans *_srf.molrep_rf. Make the third run inputting both of these peak values. Give NMON as 4. You can also use the protein sequence hare. Then refine it with Refmac, that again should decrease your R factor. I will be highly benefitted if some one point out my mistake. regards Debajyoti On Fri, 25 Jul 2008 Carl Soja wrote : Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja
[ccp4bb] convert and direction cosines
Dear, I was wondering if it is possible to preserve the direction cosines when converting an .mtz file (without scaling) to an .hkl file using the CCP4 program 'convert'..? (or perhaps another program..?) Many thanks Kristof -- Kristof Van Hecke, PhD Biomoleculaire Architectuur Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 -- Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm
[ccp4bb] Beamline scientist position EMBL-Grenoble
Staff scientist position available at the EMBL-Grenoble http://www-db.embl.de/jss/servlet/de.embl.bk.emblGroups.JobsPage/08054.html?EmblGR=x We currently looking for a beamline scientist at the EMBL-Grenoble for the mainentance, routine operation and further development of the highly successful microfocus beamline ID23-2 at the ESRF. http://www.esrf.fr/UsersAndScience/Experiments/MX/About_our_beamlines/ID23-2 For further information please contact Dr. Andrew McCarthy email: [EMAIL PROTECTED] Please send your application (cover letter, CV (in english) and contact information of three professional references) quoting ref. no. W/08/054 in the the subject line to [EMAIL PROTECTED] We are looking forward to your application, Andrew McCarthy, EMBL-Grenoble, 6, rue Jules Horowitz, 38042 Grenoble, France. Tel: +33 (0)4.76.20.72.76 08-054.pdf Description: Adobe PDF document
Re: [ccp4bb] convert and direction cosines
If you are desparate, XPREP can apply symmetry transformations to direction cosines (assuming that they are defined according to the (1976) SHELX convention, i.e. relative to the crystal reciprocal axes). 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. +49-551-39-22582 On Fri, 25 Jul 2008, Kristof Van Hecke wrote: Dear, I was wondering if it is possible to preserve the direction cosines when converting an .mtz file (without scaling) to an .hkl file using the CCP4 program 'convert'..? (or perhaps another program..?) Many thanks Kristof -- Kristof Van Hecke, PhD Biomoleculaire Architectuur Celestijnenlaan 200 F B-3001 Heverlee (Leuven) Tel: +32(0)16327477 -- Disclaimer: http://www.kuleuven.be/cwis/email_disclaimer.htm
Re: [ccp4bb] MR problem --molrep
Dear Debajyoti Thank you very much for your help. I input the each peak values as 10 and carried out the rotation and translation function again. I didn't get a improved solution by molrep. can i edit the self-rotaion function and translation function solution file as input ? best regards, carl soja Hi, I dare to say about the possible way to do molrep from my recent experience. You can choose rotation and translation function job at first and do the self rotation fuction (for multimer) after that.Each of these run will generate two different outputs *_rf.molrep_rf ans *_srf.molrep_rf. Make the third run inputting both of these peak values. Give NMON as 4. You can also use the protein sequence hare. Then refine it with Refmac, that again should decrease your R factor. I will be highly benefitted if some one point out my mistake. regards Debajyoti On Fri, 25 Jul 2008 Carl Soja wrote : Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja
Re: [ccp4bb] MR problem --molrep
Carl Soja wrote: Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja To be honest, I would try both Phaser and EPMR (now Open-EPMR) first to do MR. Both especially excel at finding good MR solutions for multimers, and are very fast as well. These programs can find solutions that are very difficult to find using other rotation-translation programs, including MOLREP. In my experience (using Phaser and EPMR) reasonable MR solutions almost always have an R-factor under 50%. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
Re: [ccp4bb] MR problem --molrep
Perhaps obvious - are you sure the space group is C222 not C2221? Phil On 25 Jul 2008, at 14:19, Roger Rowlett wrote: Carl Soja wrote: Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja To be honest, I would try both Phaser and EPMR (now Open-EPMR) first to do MR. Both especially excel at finding good MR solutions for multimers, and are very fast as well. These programs can find solutions that are very difficult to find using other rotation- translation programs, including MOLREP. In my experience (using Phaser and EPMR) reasonable MR solutions almost always have an R- factor under 50%. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
[ccp4bb] space group stats
Regarding Phil's comment about the space group, check the PBD stats and you'll see that c222 is pretty rare. It occurs only in 0.24% of all cases, versus 5.1% of C2221. So i guess you could say that without doing any analysis, there's a 95% chance that a centered orthorhombic cell is c2221 rather than c222. In terms of stats, its also pretty interesting that 58% of all structures are in only 5 (low-symmetry) space groups: p212121, p21, c2, p21212 and c2221. Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm From: CCP4 bulletin board on behalf of Phil Evans Sent: Fri 7/25/2008 9:43 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] MR problem --molrep Perhaps obvious - are you sure the space group is C222 not C2221? Phil On 25 Jul 2008, at 14:19, Roger Rowlett wrote: Carl Soja wrote: Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja To be honest, I would try both Phaser and EPMR (now Open-EPMR) first to do MR. Both especially excel at finding good MR solutions for multimers, and are very fast as well. These programs can find solutions that are very difficult to find using other rotation- translation programs, including MOLREP. In my experience (using Phaser and EPMR) reasonable MR solutions almost always have an R- factor under 50%. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
[ccp4bb] Beamline Stability Issues
Hello all! Does someone have experience in minimize energy instabilities in beamlines? MX2, our new beamline devoted to MX experiments, are facing problems with energy drifts. As far as we could notice, theses drifts are results of the contribution from several sources - possibly electron beam movements, heating of optical elements, etc... LNLS is a 2nd generation machine with 4 straight sections available for insertion devices. MX2 is a 2T wiggler-based beamline and produces a peak flux of 10^11 photons/s. What I'd like to know, before start performing calculations, how far should I expect the heating of a non-cooled 2nd crystal affects energy? Does someone know cases of a few eVs drifts? Thanks in advance and regards, Lucas Sanfelici Physicist Brazilian Synchrotron Ligth Source- LNLS (www.lnls.br) Diagnostics Group PO Box 6192 Postal Code 13083-970 Campinas-SP Brazil Phone: +55-19-3512-1153/1152 Fax: +55-19-3512-1006 E-mail: [EMAIL PROTECTED]
[ccp4bb] CCP4MG can't start
I have tried installing the newest version of CCP4MG 1.1.1, the closedirfix version, and QtMG 1.99.0. In each case, the program does not start. I did get a problem report for pyton, shown below. Mac ppc dual 2.7 GHz, OSX 10.5.4 X11= XQuartz 2.3.0(xorg-server 1.4.2-apple5) Any suggestions would be greatly appreciated! Kendall -- Kendall W. Nettles, PhD Assistant Professor Department of Cancer Biology The Scripps Research Institute 5353 Parkside Dr. Jupiter Fl 33458 office 561-799-8851 fax 561-799-8805 cell 561-306-7566 Problem report for python: Process: python [189] Path: /Applications/ccp4mg.app/Contents/MacOS/../ccp4mg-1.1.1/bin/..//pythondist// bin/python Identifier: python Version: ??? (???) Code Type: PPC (Native) Parent Process: sh [160] Date/Time: 2008-07-25 09:12:14.642 -0400 OS Version: Mac OS X 10.5.4 (9E17) Report Version: 6 Exception Type: EXC_BAD_ACCESS (SIGBUS) Exception Codes: KERN_PROTECTION_FAILURE at 0x0020 Crashed Thread: 0 Thread 0 Crashed: 0 libSystem.B.dylib0x96594494 pthread_mutex_lock + 40 1 libSystem.B.dylib0x96592e68 free + 92 2 libSystem.B.dylib0x9662cad8 closedir + 52 3 libfont_cache.dylib 0x029ae724 LoadAllFreeTypeFonts() + 4052 (freetype_font.cc:1009) 4 libfont_cache.dylib 0x029a04b0 FontCache::LoadAllFonts() + 16 (font_info.cc:251) 5 _font_cache.so 0x03a72d70 _wrap_FontCache_LoadAllFonts + 48 (font_cache_wrap_py.cc:3913) 6 org.python.python0x0016f104 _PyEval_SliceIndex + 16528 7 org.python.python0x00171360 PyEval_EvalCodeEx + 2256 8 org.python.python0x001714b0 PyEval_EvalCode + 44 9 org.python.python0x0018d534 PyErr_Display + 1932 10 org.python.python0x0018f42c PyRun_SimpleFileExFlags + 424 11 org.python.python0x001987f0 Py_Main + 1988 12 python 0x257c start + 400 13 python 0x2424 start + 56 Thread 0 crashed with PPC Thread State 32: srr0: 0x96594494 srr1: 0xd030 dar: 0x0020 dsisr: 0x4000 r0: 0x4d555458 r1: 0xbfffce40 r2: 0xa0bfcab4 r3: 0x0020 r4: 0x r5: 0x r6: 0x80808080 r7: 0x r8: 0x r9: 0x029b32cc r10: 0x6004 r11: 0xa0bfd874 r12: 0x9659446c r13: 0xbfffcfb4 r14: 0x029ad764 r15: 0xbfffd15c r16: 0x090a65c0 r17: 0x r18: 0xbfffcf78 r19: 0x r20: 0xbfffcf4c r21: 0x r22: 0x0001 r23: 0x0001 r24: 0xbfffcf74 r25: 0xbfffd168 r26: 0xbfffd16c r27: 0xbfffd170 r28: 0x0020 r29: 0xa0bf8924 r30: 0x r31: 0x9659447c cr: 0x88248204 xer: 0xlr: 0x9659447c ctr: 0x9659446c vrsave: 0x Binary Images: 0x1000 -0x2fef +python ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/bin/python 0x51000 -0x53fff +_ssl.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/_ssl.so 0x9d000 -0x9eff7 +math.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/math.so 0xa1000 -0xa2fe7 +libmginterrupt.dylib ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/lib/libmginterrupt.dylib 0xaf000 -0xb5ff7 +_socket.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/_socket.so 0xbb000 -0xbcfff +time.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/time.so 0xc -0xc1ff2 +libXau.6.dylib ??? (???) bfcdfbb3063882f751c11f520b6ac773 /usr/X11/lib/libXau.6.dylib 0x104000 - 0x1c9ff7 org.python.python 2.3.5 a (2.3.5 a) 0d8cca187f8ff8c4eacf04b69a66f39b /System/Library/Frameworks/Python.framework/Versions/2.3/Python 0x288000 - 0x28bfff +strop.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/strop.so 0x2cf000 - 0x2dbffb +libXpm.4.dylib ??? (???) aaf5463bfc482f192b8c3d23c446991b /usr/X11R6/lib/libXpm.4.dylib 0x2df000 - 0x2edfff +libXext.6.dylib ??? (???) 92d500d9cfda747c5a1c9a3d43c16104 /usr/X11R6/lib/libXext.6.dylib 0x2f9000 - 0x2fcff7 +libXdmcp.6.dylib ??? (???) cecb0b212033df7f58df772aed52bf36 /usr/X11/lib/libXdmcp.6.dylib 0x68 - 0x685ff0 +libSM.6.dylib ??? (???) f79c5f0032ecbfca21c93c55e00b2072 /usr/X11R6/lib/libSM.6.dylib 0x689000 - 0x690ff3 +libXi.6.dylib ??? (???) 6e141ddf695eb5b9732d75fb861361f0 /usr/X11R6/lib/libXi.6.dylib 0x693000 - 0x695fff +binascii.so ??? (???) /Applications/ccp4mg.app/Contents/ccp4mg-1.1.1/pythondist/lib/python2.3/lib- dynload/binascii.so 0x6d8000 - 0x713feb +_opengl.so ??? (???)
Re: [ccp4bb] CCP4MG can't start
Hi, I've had the same problem and discovered that in order to start CCP4MG the X11 must be closed. So, just quit your X11 and then start again. It worked fine for me. Andrzej Kendall Nettles wrote: I have tried installing the newest version of CCP4MG 1.1.1, the closedirfix version, and QtMG 1.99.0. In each case, the program does not start. I did get a problem report for pyton, shown below. Mac ppc dual 2.7 GHz, OSX 10.5.4 X11= XQuartz 2.3.0(xorg-server 1.4.2-apple5) Any suggestions would be greatly appreciated! Kendall
Re: [ccp4bb] MR problem --molrep
In addition to Bert's statistics argument (I've never had the pleasure of working w/ a C222 crystal), do check your self-Patterson map. I recently had a difficult MR case; the crystal masqueraded as P21212 or P2221, but it was actually P212121 with the two molecules in the AU related by a non-crystallographic translation. Dave Borhani -Original Message- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Roger Rowlett Sent: Friday, July 25, 2008 10:30 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] MR problem --molrep Good point. One should always check all the screw axis combos. Not fun for P422, but Phaser makes this very easy, as it will do all the possible screw axis combinations in one job. The correct space group and MR solution is usually very obvious if the model is acceptable and the MR parameters have been set reasonably. I think Open-EPMR will also examine screw axis combinations now, but I've never used it to do this. Cheers, Roger Rowlett Phil Evans wrote: Perhaps obvious - are you sure the space group is C222 not C2221? Phil On 25 Jul 2008, at 14:19, Roger Rowlett wrote: Carl Soja wrote: Dear all I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at first translation function. However, the second translation function Rfac is 0.526, the third is 0.525, the fourth is 0.525. All of the translation function Rfacs are too closed. I changed the model and minimum resolution for search, the Rfactor closed no any improved. My structure estmates four molecules in the aymmetric unit. This is my first time found the closed Rfac by molrep. From the low translation function Rfac, it seems that it is correct solution. I checked the solution and found some clashes in the structure.I am not sure why the Rfactor too closed in next the molecule search? I know this is unusual solution by molrep. Does it mean the diffraction data has problem? Any suggestions are welcome.Thank you in advance! Carl soja To be honest, I would try both Phaser and EPMR (now Open-EPMR) first to do MR. Both especially excel at finding good MR solutions for multimers, and are very fast as well. These programs can find solutions that are very difficult to find using other rotation- translation programs, including MOLREP. In my experience (using Phaser and EPMR) reasonable MR solutions almost always have an R- factor under 50%. Cheers, -- -- -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: [EMAIL PROTECTED]
Re: [ccp4bb] CCP4MG can't start
CCPMG is launching X11 right before it quits. Could it relate to the version of X11? Coot was not working with the version that updates with the OSX, so I had to install the Xquartz version to get Coot to work. Kendall On 7/25/08 10:53 AM, Jendrek [EMAIL PROTECTED] wrote: Hi, I've had the same problem and discovered that in order to start CCP4MG the X11 must be closed. So, just quit your X11 and then start again. It worked fine for me. Andrzej Kendall Nettles wrote: I have tried installing the newest version of CCP4MG 1.1.1, the closedirfix version, and QtMG 1.99.0. In each case, the program does not start. I did get a problem report for pyton, shown below. Mac ppc dual 2.7 GHz, OSX 10.5.4 X11= XQuartz 2.3.0(xorg-server 1.4.2-apple5) Any suggestions would be greatly appreciated! Kendall
[ccp4bb] Research associate position to work in the Membrane Protein Laboratory, Oxfordshire
Research associate position to work in the Membrane Protein Laboratory, Oxfordshire A post-doc position is available to work in the Imperial College Membrane Protein Laboratory (http://www.diamond.ac.uk/Science/MPL/default.htm) at the Diamond Light Source Synchrotron, Oxfordshire (http://www.diamond.ac.uk). The Imperial College membrane protein structures group are world leaders in crystallisation and crystallography of membrane proteins. In particular, the group has a proven track record working with membrane transporters, respiratory and photosynthetic membrane protein complexes. We are looking for a post-doc to work with Dr Liz Carpenter, Dr Bernadette Byrne and Prof. So Iwata as part of the European Drug Initiative on Channels and Transporters (EDICT: http://www.edict-project.eu/), funded by the European Union framework 7 program. The project will involve the study of metabolite/ion transporters by X-ray crystallography and the focus of the research will be potential drug targets. The research associate will be responsible for expression, purification, crystallization and X-ray crystallographic studies on a series of targets. The Membrane Protein Laboratory at Diamond is a state-of-the-art facility for crystallization and crystallography of membrane proteins, with a fully automated Thermo Fisher crystallization system and a fluidigm microfluidics system. This post is available immediately and is funded for 35 months. For informal enquiries please contact Dr Liz Carpenter ([EMAIL PROTECTED]). For more information and an application form please follow the link below: http://www3.imperial.ac.uk/employment/research/ns2008105ab Please send applications and your cv to Mrs Mutsuko Grant ([EMAIL PROTECTED]) before the closing date on the 5th of August, 2008.
Re: [ccp4bb] CCP4MG can't start
Hi Kendall: I haven't used CCP4Mg^2+ but if it is launched by a shell script that dumbly sets the DISPLAY variable, then on 10.5 this is equivalent to a suicide directive, since the new X11 (as of 10.5) sets the DISPLAY variable using launchd. If this is the case, hack that line out of the script or put in a conditional test for the operating system version. The latest X11 for 10.5.4 is worth getting too, as it has a lot of improvements: http://xquartz.macosforge.org/trac/wiki/X112.3.0 Bill William G. Scott Contact info: http://chemistry.ucsc.edu/~wgscott/ On Jul 25, 2008, at 9:18 AM, Kendall Nettles wrote: CCPMG is launching X11 right before it quits. Could it relate to the version of X11? Coot was not working with the version that updates with the OSX, so I had to install the Xquartz version to get Coot to work. Kendall On 7/25/08 10:53 AM, Jendrek [EMAIL PROTECTED] wrote: Hi, I've had the same problem and discovered that in order to start CCP4MG the X11 must be closed. So, just quit your X11 and then start again. It worked fine for me. Andrzej Kendall Nettles wrote: I have tried installing the newest version of CCP4MG 1.1.1, the closedirfix version, and QtMG 1.99.0. In each case, the program does not start. I did get a problem report for pyton, shown below. Mac ppc dual 2.7 GHz, OSX 10.5.4 X11= XQuartz 2.3.0(xorg-server 1.4.2-apple5) Any suggestions would be greatly appreciated! Kendall
[ccp4bb] ccp4 6.0.99e test release
Dear All the latest test version is on the ccp4 ftp server. ftp://ftp.ccp4.ac.uk/ccp4/6.1/ccp4-6.0.99e-core-src.tar.gz- core ccp4, rapper, clipper ftp://ftp.ccp4.ac.uk/ccp4/6.1/ccp4-6.0.99e-phaser-src.tar.gz - cctbx and phaser ftp://ftp.ccp4.ac.uk/ccp4/6.1/ccp4-6.0.99e-balbes_db.tar.gz - balbes database only There is also the dependency of PyXML if you want to run balbes ftp://ftp.ccp4.ac.uk/ccp4/6.1/ccp4-6.0.99e-PyXML.tar.gz for the first 3 tarballs unpack into the same directory, then configure...make...make install. For the PyXML, follow the instructions in the PyXML-0.8.4 directory. Major changes: refmac5.5 built by default. This gives twinning and sad refinement. dbhandler. Many optimisations, so this should be much more responsive. For other updates see the CHANGES file. Still to come: downloads pages (under internal testing). documentation updates (lots of) Feedback to the usual locale ([EMAIL PROTECTED]) Thanks Charles and the rest of us here at DL. -- *** * * * Dr. Martyn Winn * * * * STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K. * * Tel: +44 1925 603455E-mail: [EMAIL PROTECTED] * * Fax: +44 1925 603825Skype name: martyn.winn * * URL: http://www.ccp4.ac.uk/martyn/ * ***
[ccp4bb] Slightly off-topic - silica based columns
Slightly off-topic - but these days most crystallographers are interested in protein purification. What experience do people have with silica based columns for size exclusion chromatography. We are interested in resolution, stability, column life time, and ease of cleaning column compared to Superdex and Superose columns. Please describe specific columns if you can. Thanks -- Mark Mayer
[ccp4bb] Refinement problem
Dear all, Now I'm solving a structure with 1.6A resolution. The data seems good with R-sym (12.4) and all other parameters. Actually the data was collected with SAD phasing. When we checked the data we couldn't find the Se atom in the structure. Since the data resolution is good, we tried to do molecular replacement using Balbes program. It was selected a model with 25% sequence identity and we got the good solution too. I could find all residues in the density and also checked the Ramachandran map which shows almost all residues are in the allowed region. The problem is, I have done refinement many times, the R-factor (45.3) and R-free (51.4) is not reducing during the refinement and also figure of merit is not increasing. Still it remains what I got during the first refinement. The density is also not improving much. Also I could find many cuts in the density. My question is…….. 1.Can we use SAD phasing data for MR solution? 2.Is there any other way to reduce the R/R-free? 3.Why the figure of merit is not increasing even after modeled the residues exactly into the electron density? Thanks, Regards, Sampath
[ccp4bb] Baculovirus expression and purification - off topic
Dear All: I have a peculiar problem with baculovirus expression of my protein. The native protein elutes in Tris gradient. This has no problems. However, a mutant elutes in the wash. After further purification with size exclusion, I notice that this mutant is always associated with some medium component that I cannot separate. I know this (?) because when lyophllized (not a sin for this protein), I get 15-30 mg of protein (or colorless fluffy material) whereas protein estimation gives only about 5 mg. This amount is what I usually get for the native protein. I have tried active charcoal, BioRex, ammonium sulfate precipitation and even hydroxyapatite columns but nothing seems to work to separate the extra material from the protein. Any insight would be of much help. Thanks a lot Subbu
Re: [ccp4bb] Refinement problem
Hi Sampath, You are asking many questions at once. Since I am right now trying to solve a very difficult Se-Met structure, here are some ideas: - Do you have an energy scan on your crystal, showing that there is absorbance at the correct wavelength for Se? If yes, you have proof that there was indeed Se in your crystal; - You describe that you cannot find the Se atom. In theory it is possible that the atom is in the crystal, but not in an ordered fashion and therefore you would not be able to find it. That is theory, I think that in the majority of cases it works fine. You have not told us how the data were collected (synchrotron? wavelength? Inverse beam protocol to optimize SAD?) and whether or not a statistical analysis (with scaleit) tells you if there is a signal there. If you see a signal, then you know Se is present AND ordered. - If you have a MR solution, you can try to use those phases to find the Se atom in a phased anomalous difference map and then (provided that everything is consistent) use a combination of experimental and MR phases; but... - You also did not tell us how you know that your MR solution is correct. Specifically, can you see any features in the structure that are not part of the search model and are sensible? If yes, your solution is useful. If no, you should try omit maps to convince yourself that the MR solution isn't (too) biased and in fact correct - Your statistics given suggest that the MR solution is very poor, with a cons iderable chance that it is not valid. Remember that ~55% R-factor is equivalent to a random solution. If you do refinement and there is no improvement and the statistics are poor, chances are good that the solution wasn't correct in the first place. I worry that you have concluded that since the model fits the density nicely, based on MR, then things must be good, but if you look at Kevin Cowtan's web page with the duck and cat, you will be reminded of the fact that with phases from MR you (almost?) always get nice density from MR, but that does not mean at all that it is correct density. So yes, you should pursue the SAD phases. Remember that the signal will be weak (we cannot judge how weak, depends on the number of Se and size of your problem and the wavelength used), but (good news) you should not have problems with non-isomorphism (since you are not comparing two data sets). The SAD phases, after appropriate density modification, may show you a partial structure - I just tried this for my problem and it did not work for me, but then again, you must try to see if it can be done. Also, assuming that you have native, S-containing protein (as opposed to Se) there is the option of comparing the S- versus Se-protein and you might be able to get phases from that. Finally, others on this forum are better in this matter: 1.6A is fairly high resolution and I wonder if it is possible to pursue direct methods. Probably too low resolution, but I wouldn't know all that w ell, my maps are 5A resolution, so I don't worry about that option. Mark -Original Message- From: Sampath Natarajan [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Sent: Fri, 25 Jul 2008 11:05 am Subject: [ccp4bb] Refinement problem Dear all, Now I'm solving a structure with 1.6A resolution. The data seems good with R-sym (12.4) and all other parameters. Actually the data was collected with SAD phasing. When we checked the data we couldn't find the Se atom in the structure. Since the data resolution is good, we tried to do molecular replacement using Balbes program. It was selected a model with 25% sequence identity and we got the good solution too. I could find all residues in the density and also checked the Ramachandran map which shows almost all residues are in the allowed region. The problem is, I have done refinement many times, the R-factor (45.3) and R-free (51.4) is not reducing during the refinement and also figure of merit is not increasing. Still it remains what I got during the first refinement. The density is also not improving much. Also I could find many cuts in the density. My question is…….. 1. Can we use SAD phasing data for MR solution? 2. Is there any other way to reduce the R/R-free? 3. Why the figure of merit is not increasing even after modeled the residues exactly into the electron density? Thanks, Regards, Sampath
Re: [ccp4bb] Refinement problem
From: Ta Hai Sent: Sat 7/26/2008 3:00 AM To: Sampath Natarajan Subject: RE: [ccp4bb] Refinement problem With the R and freeR factor 45.3% and 51.4%, I'm quite sure that your MR solution is not correct. Although your current model seems fitting with the map, but it's just the biased map after refinement with REFMAC. While now you can't be sure that whether your crystal contain Se atom or not, try MR again: - Make sure you estimate correctly the numbers of molecules/AU. - Prepare the best search model as much as you can - Try with different programs like Molrep, EPMR, Phaser... Good luck, Hai From: CCP4 bulletin board on behalf of Sampath Natarajan Sent: Sat 7/26/2008 2:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Refinement problem Dear all, Now I'm solving a structure with 1.6A resolution. The data seems good with R-sym (12.4) and all other parameters. Actually the data was collected with SAD phasing. When we checked the data we couldn't find the Se atom in the structure. Since the data resolution is good, we tried to do molecular replacement using Balbes program. It was selected a model with 25% sequence identity and we got the good solution too. I could find all residues in the density and also checked the Ramachandran map which shows almost all residues are in the allowed region. The problem is, I have done refinement many times, the R-factor (45.3) and R-free (51.4) is not reducing during the refinement and also figure of merit is not increasing. Still it remains what I got during the first refinement. The density is also not improving much. Also I could find many cuts in the density. My question is 1.Can we use SAD phasing data for MR solution? 2.Is there any other way to reduce the R/R-free? 3.Why the figure of merit is not increasing even after modeled the residues exactly into the electron density? Thanks, Regards, Sampath
[ccp4bb] RES: [ccp4bb] Beamline Stability Issues
Hey Andrew! Thanks for answering! Does someone have experience in minimize energy instabilities in beamlines? Are you sure it's an energy instability and not an intensity one? It's quite rare to see energy instabilities from a well calibrated monochromator. All monochromators should be in a closed loop, if there really are energy instabilities, then it's either mechanical (the encoder or crystal is loose) or the closed loop parameters are wrong for the rotational motor and the mono is slipping out of the closed loop window. Yes, I'm sure. I'm certain most of this problem arises from thermal issues in the monochromator. For example, we control the temperature of our 1st crystal, but for the 2nd one there is kind of control, the tunning between them is not closed-looped through a MOSTAB or something, there is no kind of shielding for the Huber. Thus, I have several reasons to be concerned! MX2, our new beamline devoted to MX experiments, are facing problems with energy drifts. As far as we could notice, theses drifts are results of the contribution from several sources - possibly electron beam movements, heating of optical elements, etc... Could well be, but these will result in intensity instabilities as opposed to energy ones Yes, this is evident in my data. LNLS is a 2nd generation machine with 4 straight sections available for insertion devices. MX2 is a 2T wiggler-based beamline and produces a peak flux of 10^11 photons/s. What I'd like to know, before start performing calculations, how far should I expect the heating of a non-cooled 2nd crystal affects energy? Does someone know cases of a few eVs drifts? The second crystal will have no effect on energy, it will have a major impact on intensity. We had extreme difficulties in stabilising and sheilding our Khozu second crystal from thermal drifts. We eventually gave up and installed a channel cut mono. The much simplier design is far more easy to work with Luck you! I'm in the start of the journey! I guess I'll have to try fix that first! :( Andrew, what kind of approach did you try to stabilize 2nd crystal? If you know someone else who could contribute in someway to this problem, please tell me. Thank you one more time, LS
Re: [ccp4bb] SUMMARY: synchrotron remote data collection
A start is made, if somebody would like to comment / add information to the open ? that would be great. http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Synchrotrons Jürgen - Jürgen Bosch University of Washington Dept. of Biochemistry, K-426 1705 NE Pacific Street Seattle, WA 98195 Box 357742 Phone: +1-206-616-4510 FAX: +1-206-685-7002 Web: http://faculty.washington.edu/jbosch
[ccp4bb] Beamline Stability Issues
Dear Mark, thanks for your answer! Yes, there is an actual change in energy and I guess my problem does not have a single source! In the case you know someone who faced/have faced a similar problem around please tell me. Brazilian regards, LS. 1. Is the energy drift a change in flux or actual change in wavelength? In the case of a change in flux it could be that you require more cooling, what temperature is the cooling water at and how constant is the water temperature? You may need to lower the cooling water temperature. On beamline 10 at the SRS, the 1st mirror is cooled by the synchrotron deionised water supply at a temperature of ~20oC, however we cool the monochromator crystals on an independent water supply, typically at a temperature of 4oC. We do see a drastic shift in beam intensity when we go to wavelengths 2.2A (beam fluctuates wildly), consequently we limit operations to wavelengths between 0.875 and 2.1A. If it is a real change in energy/wavelength then there is a more serious problem. It would suggest a change in the beam entering the optical elements.
[ccp4bb] Meaning of sigma level of electron density map?
I am new to the filed of crystallography. I am having trouble figuring out what exactly does sigma level of electron density map mean. When sigma level of a map is increased (say from 1.5 sigma to 2 sigma) why the map covering individual residues becomes less wide and more precise? Shouldn't it be the other way round if they have anything to do with the Gaussian districution? Thanks!
Re: [ccp4bb] Meaning of sigma level of electron density map?
P K wrote: I am new to the filed of crystallography. I am having trouble figuring out what exactly does sigma level of electron density map mean. When sigma level of a map is increased (say from 1.5 sigma to 2 sigma) why the map covering individual residues becomes less wide and more precise? Shouldn't it be the other way round if they have anything to do with the Gaussian districution? Dear Great Biologist, Imagine starting on the plains of Tanzania and walking up Mt Kilimanjaro. Every 100 steps you make an note of your elevation. You get to the top and make your last note. You come down again. You decide to do some statistics on your elevation data (relative to you starting position). You calculate a mean and standard deviation. Questions the you might like to ask yourself at this point: do the observations correspond to a Gaussian distribution? Does that matter? The next day you walk back up again. You go to the elevation corresponding to 1.5 sigma. You take a piece of string and on your map, you trace round the contour level that corresponds to your current elevation. Question: What is the length of that piece of string? You walk a bit further (to 2 sigma, say). You again get out your map and string. Question: Is the length of string longer or shorter than it was previously? Is your position any more precise than it was earlier? Is that a sensible question? Shouldn't the relative lengths be the other way round if they have anything to do with the Gaussian distribution? I hope that clears things up a bit.
[ccp4bb] offtopic-colorful tag
Hi all, I am curious whether there is an expression system in which the target protein will be expressed with a tag, which has a strong extinction coefficient in visible wavelength range? That will dramatically simplify my protein purification and crystallization. Thanks for your suggestion. Deliang
Re: [ccp4bb] offtopic-colorful tag
Those colorful tags may be too big for crystallization purpose. You can try GFP or heme based tags. A few companies are promoting heme based red tags. Red color is not very intense. You'd be better off with UV or GFP. There are small molecule tags that links to Cys. But you'll spend a fortune to get enough to see. They are Ni-NTA linked dyes as well. If your protein expresses well, you can see it while you growing the culture. Chun Chun Luo, Ph.D. The Protein Expert Accelagen, Inc. 11585 Sorrento Valley Road, Suite 107 San Diego, CA 92121 TeL: 858-350-8085 ext 111 Fax: 858-350-8001 mailto:[EMAIL PROTECTED] [EMAIL PROTECTED] www.accelagen.com http://www.accelagen.com/ _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of deliang Sent: Friday, July 25, 2008 4:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] offtopic-colorful tag Hi all, I am curious whether there is an expression system in which the target protein will be expressed with a tag, which has a strong extinction coefficient in visible wavelength range? That will dramatically simplify my protein purification and crystallization. Thanks for your suggestion. Deliang
[ccp4bb] postdoc positions availabel
Postdoctoral positions available immediately in a membrane transport and structural biology laboratory. Our new laboratory in the Department of Cell Physiology and Molecular Biophysics at Texas Tech University Health Science Center at Lubbock is well equipped with the state-of the-art equipment for robotic high throughput membrane protein crystallization. Our current focus is on crystallization and structural determination of bacterial and human sugar transporters. Highly motivated candidates with a strong background either in protein X-ray crystallography, or in biochemistry and biophysics are encouraged to apply. More information is available at http://www.ttuhsc.edu/SOM/physiology/faculty/Guan/guan.aspx. Salary is competitive based on experience. Please submit a CV and contact information for three references to Dr. Lan Guan through TTUHSCs website (http://jobs.texastech.edu), requisition #77090 or #77091. Lan Guan Assistant Professor Department of Cell Physiology Molecular Biophysics Texas Tech University Health Sciences Center 3601 4th Street, STOP 6551 Lubbock, Texas 79430 Email: [EMAIL PROTECTED] http://www.ttuhsc.edu/SOM/physiology/faculty/Guan/guan.aspx.
[ccp4bb] question about getting rid of model bias in refinement
Hello Everyone, I have a question about getting rid of model bias in refinement with refmac. I solved the structure with molecular replacement. After final refinement of the structure, I found out some key amino acids in the structure and wanted to make sure their conformations are correct. I omitted these amino acids (by setting occupancy to zero) and refined the structure. I manually fit the amino acids into the density and refined the structure again. I found these amino acids return to the precious conformations even though the conformations I fit were different. Should I omit these amino acids from the beginning of the refinement? What is the best way to get rid of the model bias? Your suggestions are greatly appreciated! Best, Sun
