Re: [ccp4bb] Heavy atom sites
Hello David, I would use the SAD target function of refmac5 for the anomalous occupancy. As of isomorphous occupancy and phasing power, I don't know. Best, Tim On 12/15/2013 10:29 PM, David Schuller wrote: I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
[ccp4bb] AW: [ccp4bb] Wrong Space Group?
Dear Bonsor, I fully second James suggestions but have a few additional comments: If you get a solution in P6522 with one molecule, you should get the same solution in P65 with 2 molecules. One of the crystallographic symmetry operators would then be non-crystallographic. The current version of Refmac will test all possible twinning operations, so there is no need to do it yourself (provided of course that you get a molecular replacement solution). I would also try your rebuilt model with extended helix as a model for MR. I suspect that the dimer which has formed is asymmetric and that it may be randomly packed in your crystal. If the helix is a small compared to the complete protein, it may not show up in twinning tests. Good luck! Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von James Holton Gesendet: Sonntag, 15. Dezember 2013 23:29 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] Wrong Space Group? Its possible you are in a lower space group, perhaps with some twinning, but your search model is different enough to only find a solution when things are over-merged. Try refining your P6522 model against data merged in P65. If the other copy (symmetry mate in P6522) does not show up, you may be in trouble (wrong MR solution). I'd also try refinement/building in the other triogonal/hexagonal space groups, but again, start with the PDB file that you got for P6522. Just change the space group in the header, and switch out the MTZ file. You will need to merge your data in each space group and also check the a-b flip re-indexing for most of them. Have a look at the CCP4 reindexing list for the h,k,l operators to try: http://www.ccp4.ac.uk/html/reindexing.html note how similar they are to the twinning operators: http://www.ccp4.ac.uk/html/twinning.html If I have counted right, that means you have 36 jobs to run. I'd also recommend turning the TWIN option in refmac off and on for each of these cases. This will always give you a lower R factor, because of the dynamic range compression you get with twinning, but if one particular combination of twinning with a particular space group and axis reindexing is markedly better than all the others, then you have just found your right space group. So, now we are up to 72 jobs, but hardly a lot of work compared to growing the crystals in the first place. You might also want to try being clever and generating the symmetry mates of your P6522 model and refine these partners as separate molecules as you reduce the symmetry of the data. It's tricky, but think of it as an exercise. Which real-space operator becomes what reciprocal-space operator? You can check your answer by loading it up in coot and seeing if symmetry mates clash with the input coordinates. Yes, its a lot of work to try all these combinations, but that's the annoying thing about twinning, it opens up a lot of ambiguities. Good luck! -James Holton MAD Scientist On 12/14/2013 6:44 AM, D Bonsor wrote: Dear all, I have collected ~160 degrees of data on a new crystal form of a protein which has already been solved. Data was processed with XDS and reindex, scaled and truncated with Aimless. Both XDS and Pointless suggested a Laue group of P6/mmm with a possible space group of P6122 or P6522. Stats showed an overall Rmerge of 0.131 but an Rpim of 0.041 (multiplicity/redundancy of 19.1), a completeness of 99.1% and resolution of 2.8Ang. With cell dimensions of 63.1 63.1 243, only one protein chain can be found in the asymmetric unit (two copies would leave a solvent content of 8%). I ran phaser with all alternative space groups and a single solution in P6522 with a TFZ of 10.0. I then performed 20 Refmac cycles ending up with an R/Rfree of 35.5/45.5. I open the structure and map in Coot and could see that there was a large conformational change of helix-turn-helix actually becoming just a long helix (https://www.dropbox.com/s/4s6g8apatsi5xcg/Before_Building.png) and then dimerizing through the long helix with one of the symmetry mates. This section was rebuilt (https://www.dropbox.com/s/5j7tv0i5yq3mxxx/After_Building.png) and ran through Refmac again resulting in an R/Rfree of 35.5/44.3. Looking through the rest of the structure I see nothing else really to be modeled. Nothing that could bring the Rfactors down to a reasonable range. I have therefore tried several things. I ran the structure through Zanuda server to look at other space group possibilities. The server suggested I was in the correct space group. However I did reprocess the data to P6, P3, P312, P321, C2221, P2 and C2, and reran phaser in search all alternative space groups using the original search model but found no solutions. I did reprocess the data in P1, though I did not collect enough data. Twinning tests show no twinning. Although that does not mean there is no twinning, I can
[ccp4bb] Postdoctoral positions in structural biology/ cryo-EM at OIST
Dear structural biologists, there are two positions for postdoctoral researchers available with an emphasis on high-resolution cryo-EM at OIST (Okinawa Institute of Science and Technology Graduate University, Japan). We are a highly motivated young international team and benefit from an excellent infrastructure which is hard to match elsewhere. * Postdoctoral position for an expert in cryo-EM * Postdoctoral position in Structural Biology http://www.oist.jp/careers The fully funded positions are available immediately and will be open until suitable candidates are identified. Best regards, Matthias ___ Matthias Wolf, PhD MPharm - Assistant Professor Molecular Cryo-Electron Microscopy Unit Okinawa Institute of Science and Technology Graduate University 1919-1 Tancha, Onna-son, Kunigami-gun Okinawa 904-0412, Japan
[ccp4bb] AW: [ccp4bb] Phaser output problem
Looks like you need to search for more molecules. Best, Herman Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Amanda Blythe Gesendet: Montag, 16. Dezember 2013 11:46 An: CCP4BB@JISCMAIL.AC.UK Betreff: [ccp4bb] Phaser output problem I am trying to solve a structure by molecular replacement using Phaser. The pdb fits the density really well but the output shows strange crystal packing. I've attached an image, does anyone know what is causing this? [cid:image001.jpg@01CEFA56.DCDA5270] Amanda PhD student Laboratory 4.26, Bayliss Building School of Chemistry and Biochemistry M313 The University of Western Australia 35 Stirling Highway Crawley WA 6009 Australia T (+61 8) 6488 3163 E lewis...@student.uwa.edu.aumailto:lewis...@student.uwa.edu.au inline: image001.jpg
Re: [ccp4bb] AW: [ccp4bb] Phaser output problem
Things to check - number of molecules to search for. You can use Matthewscoeff to get a suggestion - MOLREP does it autromatically . Space group - has Phaser chosen as best SG an alternative to that in your header? Eleanor On 16 December 2013 11:03, herman.schreu...@sanofi.com wrote: Looks like you need to search for more molecules. Best, Herman *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von *Amanda Blythe *Gesendet:* Montag, 16. Dezember 2013 11:46 *An:* CCP4BB@JISCMAIL.AC.UK *Betreff:* [ccp4bb] Phaser output problem I am trying to solve a structure by molecular replacement using Phaser. The pdb fits the density really well but the output shows strange crystal packing. I've attached an image, does anyone know what is causing this? Amanda PhD student Laboratory 4.26, Bayliss Building School of Chemistry and Biochemistry M313 The University of Western Australia 35 Stirling Highway Crawley WA 6009 Australia *T* (+61 8) 6488 3163 *E* lewis...@student.uwa.edu.au image001.jpg
Re: [ccp4bb] Heavy atom sites
I would find the sites from the PHIC - you need to use CAD to add Fcalc PHIC and FOM to the original data with Fnative Fderiv DANOderiv etc I usually then use SCALEIT to scale native and derivative to Fcalc - then you know you are roughly on an absolute scale Then feed those sites into Phaser_EP and it will refine occupancy etc and give you phasing power Eleanor On 16 December 2013 09:21, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Hello David, I would use the SAD target function of refmac5 for the anomalous occupancy. As of isomorphous occupancy and phasing power, I don't know. Best, Tim On 12/15/2013 10:29 PM, David Schuller wrote: I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
[ccp4bb] ACA 2014, Industrial Research from Young Scientists
The Industrial Special Interest Group and Young Scientists Special Interest Group of the American Crystallographic Association will be hosting a scientific session on Sunday, May 25, 2014 during the ACA Meeting in Albuquerque, New Mexico which will highlight research by young scientists (undergraduate, graduate, post-doc) that is done in industry or in collaboration with industry. We encourage young scientists in this area to submit an abstract for consideration for a talk during this session. A brief description of the session is highlighted below,1.2.1 Industrial Research from Young ScientistsOrganizers: George Lountos, Pete Wood An industrial environment can provide invaluable research and career experience for students and other young scientists. At the same time a short or long term research placement can provide an industrial partner with the opportunity to have very focussed research carried out in an otherwise time-stretched setting. This session will showcase research being performed by young scientists at any level (undergraduate, post-graduate or early post-doctoral) either within industry or funded by industry. The deadline for abstract submission is January 31, 2014. Abstracts may be submitted online at http://www.amercrystalassn.org/2014-abstractsYoung scientists wishing to present at the meeting are also encouraged to apply for a Travel Grant. http://www.amercrystalassn.org/2014-young-scientistsIf you have any questions, feel free to contact the session organizers, Pete Wood (w...@ccdc.cam.ac.uk) or George Lountos (lount...@mail.nih.gov)Thanks,George
[ccp4bb] CAD
Dear all, I am trying to use CAD to apply b-factor sharpening to my 3.7 A data. Following is the script which I am running. cad hklin1 test.mtz hklout test_out.mtz END-cad RESOLUTION OVERALL 40 3.7 scale file_number 1 1 -50 END END-cad test.mtz is the refmac output. The output fails to open in Coot. Could any please correct the script? Thanks, Ashu
Re: [ccp4bb] CAD
Hi, It looks like you're missing your LABIN line. Pete Ashu Kumar wrote: Dear all, I am trying to use CAD to apply b-factor sharpening to my 3.7 A data. Following is the script which I am running. cad hklin1 test.mtz hklout test_out.mtz END-cad RESOLUTION OVERALL 40 3.7 scale file_number 1 1 -50 END END-cad test.mtz is the refmac output. The output fails to open in Coot. Could any please correct the script? Thanks, Ashu
Re: [ccp4bb] ccp4 v6.4.0 cannot extract mtz
Dear Uli, On Mon, 2013-12-16 at 15:52 +, ulrich.goh...@mdc-berlin.de wrote: Dear colleagues, Trying to run the latest version of ccp4 (6.4.0), say Refmac, I get the following error when I fill in the file box for the data file: CCP4i encountered an error when trying to extract the data from data.mtz. You can view the output from the mtzdump program etc. + crash of the GUI. Now, running mtzdump gives me this: ./mtzdump: /usr/lib64/libgfortran.so.3: version `GFORTRAN_1.1' not found (required by /xprogs/CCP4/CCP4-6.4.0/destination/ccp4-6.4.0/bin/../lib/libccp4f.so.0) In /usr/lib64 I have softlinked libgfortran.so.3 - libgfortran.so.3.0.0 (did so in 2011). I am not sure what I need to update here and how. I am running Novell's SLES 11 on a 64 bit system with the libgfortran43 runtime library installed. I have also checked file permissions (incl. tmp directories), and I cannot find anything obvious. I cannot remember having had similar problems with v6.3.0 in the beginning. This may be a problem with the version information in the gfortran library supplied by SUSE not containing anything matching the version used by CCP4 on the system that they use for building their binaries. For more information, you could try the commands: ldd -v /path/to/mtzdump which will list explicitly the version information of the required shared libraries, and: readelf -V /usr/lib64/libgfortran.so.3 and looking for the section starting Version definition section: it should look something like this: Version definition section '.gnu.version_d' contains 9 entries: Addr: 0x00015da0 Offset: 0x015da0 Link: 5 (.dynstr) 00: Rev: 1 Flags: BASE Index: 1 Cnt: 1 Name: libgfortran.so.3 0x001c: Rev: 1 Flags: none Index: 2 Cnt: 1 Name: GFORTRAN_1.0 0x0038: Rev: 1 Flags: none Index: 3 Cnt: 2 Name: GFORTRAN_1.1 0x0054: Parent 1: GFORTRAN_1.0 0x005c: Rev: 1 Flags: none Index: 4 Cnt: 2 Name: GFORTRAN_1.2 0x0078: Parent 1: GFORTRAN_1.1 0x0080: Rev: 1 Flags: none Index: 5 Cnt: 2 Name: GFORTRAN_1.3 0x009c: Parent 1: GFORTRAN_1.2 0x00a4: Rev: 1 Flags: none Index: 6 Cnt: 2 Name: GFORTRAN_1.4 0x00c0: Parent 1: GFORTRAN_1.3 0x00c8: Rev: 1 Flags: none Index: 7 Cnt: 1 Name: F2C_1.0 0x00e4: Rev: 1 Flags: none Index: 8 Cnt: 1 Name: GFORTRAN_C99_1.0 0x0100: Rev: 1 Flags: none Index: 9 Cnt: 2 Name: GFORTRAN_C99_1.1 0x011c: Parent 1: GFORTRAN_C99_1.0 I suspect that your version may be missing the GFORTRAN_1.1 entry (the above listing is from openSUSE 12.3, which does have the correct version). If so, maybe this is something that CCP4 need to deal with in their build of the official binaries. Alternatively, if you build CCP4 from source on your system, it should work. Has anyone encountered the same problem and solved it yet? We have a SLES licence at Global Phasing, although we haven't run CCP4 on a SLES system for a long time now. Your experience suggests that we should give it a try again: we had been assuming that if it works with openSUSE then SLES/SLED should also be OK but now it seems not :-) I'm afraid that I am unlikely to have time to do this before January though, so it won't help you if you are in a hurry. Unless someone else who is running CCP4 under SLES has a quick fix, I think that building CCP4 from source may be your quickest solution. Regards, Peter. -- Peter Keller Tel.: +44 (0)1223 353033 Global Phasing Ltd., Fax.: +44 (0)1223 366889 Sheraton House, Castle Park, Cambridge CB3 0AX United Kingdom
Re: [ccp4bb] MrBUMP not running after update
Dear Jie, Thanks for spotting this problem. I've made a fix for MrBUMP that should address the problem. It will be included in the next CCP4 update which will go out tomorrow (Tuesday 17th of December) all being well. Best wishes, Ronan On 10/12/13 01:34, jie liu wrote: Dear All I just installed CCP4-6.4 and all the subsequent updates. Now MrBump failed with the following error message: The program run with command: /usr/local/ccp4-6.4.0/bin/ccp4-python -u /usr/local/ccp4-6.4.0/bin/mrbump HKLIN /home/jie/Structures/local/se-p26c8newn6/ccp4_6.3/high_x4c.mtz SEQIN /home/jie/Structures/local/se-p26c8newn6/ccp4_6.3/seq.seq HKLOUT /home/jie/Structures/local/se-p26c8newn6/ccp4_6.3/high_x4c_mrbump_soln1.mtz XYZOUT /home/jie/Structures/local/se-p26c8newn6/ccp4_6.3/high_x4c_mrbump_soln1.pdb has failed with error message Traceback (most recent call last): File /usr/local/ccp4-6.4.0/bin/mrbump, line 107, in module queue = master.fast_search_MR(init, target_info, mstat) File /usr/local/ccp4-6.4.0/share/mrbump/include/initialisation/MRBUMP_master.py, line 482, in fast_search_MR ps.get_top_hit(mstat.sorted_MR_list, init, mstat) File /usr/local/ccp4-6.4.0/share/mrbump/include/structures/Matches.py, line 1431, in get_top_hit CHAIN=mstat.chain_list[chain] KeyError: '1e6j_p2' It was running fine with CCP4-6.3. Could someone help please? Thanks! Jie -- Scanned by iCritical.
Re: [ccp4bb] ccp4 v6.4.0 cannot extract mtz
Yes Uli. I have the same issue, but not solve it yet :-( Partha On Mon, Dec 16, 2013 at 10:52 AM, ulrich.goh...@mdc-berlin.de ulrich.goh...@mdc-berlin.de wrote: Dear colleagues, Trying to run the latest version of ccp4 (6.4.0), say Refmac, I get the following error when I fill in the file box for the data file: CCP4i encountered an error when trying to extract the data from data.mtz. You can view the output from the mtzdump program etc. + crash of the GUI. Now, running mtzdump gives me this: http://www.hkl-xray.com/data-upload-form-and-bug-report./mtzdump: /usr/lib64/libgfortran.so.3: version `GFORTRAN_1.1' not found (required by /xprogs/CCP4/CCP4-6.4.0/destination/ccp4-6.4.0/bin/../lib/libccp4f.so.0) In /usr/lib64 I have softlinked libgfortran.so.3 - libgfortran.so.3.0.0 (did so in 2011). I am not sure what I need to update here and how. I am running Novell's SLES 11 on a 64 bit system with the libgfortran43 runtime library installed. I have also checked file permissions (incl. tmp directories), and I cannot find anything obvious. I cannot remember having had similar problems with v6.3.0 in the beginning. Has anyone encountered the same problem and solved it yet? My apologies if this has been discussed already; I searched the archive and I couldn't find a similar thread. Cheers, Uli --- dr ulrich gohlke *staff scientist - m**acromolecular structure and interaction* max-delbrück-center for molecular medicine (mdc) +49 30 9406 - 2725 (w) +49 30 9406 - 2548 (fax) ulrich.goh...@mdc-berlin.de http://www.mdc-berlin.de/en/research/research_teams/macromolecular_structure_and_interaction/
Re: [ccp4bb] Heavy atom sites
Didn't mlphare use to print those values in the log file ? Jürgen On Dec 15, 2013, at 4:29 PM, David Schuller dj...@cornell.edumailto:dj...@cornell.edu wrote: I have some SIRAS data of a known structure. I want to get the isomorphous and anomalous occupancy and phasing power from my data. What's the best software to do this? -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edumailto:schul...@cornell.edu .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu
[ccp4bb] Cryo solution for crystals grown in magnesium formate
Dear all, sorry if this topic does not interest you. I wonder whether anyone has experience with freezing crystals grown in ~0.2 M Magnesium Formate. Garman and Mitchell suggested that A major anomaly is solution 44, 0.2 M magnesium formate, which requires 50% glycerol for cryoprotection in their 1996 paper (J Appl. Cryst. 29, 584-587). Since 50% glycerol is kind of harsh, I wonder whether anyone has tried alternative cryo protectant. Your kind help will be highly appreciated. Best regards, Junyu --- Junyu Xiao, Ph.D. University of California, San Diego Leichtag Room 283 9500 Gilman Drive, 0721 La Jolla, CA 92093-0721 Lab phone: 858-822-0684
Re: [ccp4bb] Cryo solution for crystals grown in magnesium formate
Junyu, I haven't tried it personally with this particular solution, but I have found that 30% glucose can pretty much cryoprotect any condition I have tried it with. If necessary, add cryoprotectant solution (mother liquor + 30% glucose) gradually to minimize osmotic shock and potential cracking of crystals. You can try this without crystals to see if the solution vitrifies as a clear solid in liquid nitrogen. If it freezes clear, it is very likely to work fine with your crystals. Cheers, ___ Roger S. Rowlett Gordon Dorothy Kline Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 12/16/2013 4:36 PM, Xiao, Junyu wrote: Dear all, sorry if this topic does not interest you. I wonder whether anyone has experience with freezing crystals grown in ~0.2 M Magnesium Formate. Garman and Mitchell suggested that A major anomaly is solution 44, 0.2 M magnesium formate, which requires 50% glycerol for cryoprotection in their 1996 paper (J Appl. Cryst. 29, 584-587). Since 50% glycerol is kind of harsh, I wonder whether anyone has tried alternative cryo protectant. Your kind help will be highly appreciated. Best regards, Junyu --- Junyu Xiao, Ph.D. University of California, San Diego Leichtag Room 283 9500 Gilman Drive, 0721 La Jolla, CA 92093-0721 Lab phone: 858-822-0684
Re: [ccp4bb] Cryo solution for crystals grown in magnesium formate
On Mon, Dec 16, 2013 at 1:36 PM, Xiao, Junyu jx...@mail.ucsd.edu wrote: Dear all, sorry if this topic does not interest you. I wonder whether anyone has experience with freezing crystals grown in ~0.2 M Magnesium Formate. Garman and Mitchell suggested that A major anomaly is solution 44, 0.2 M magnesium formate, which requires 50% glycerol for cryoprotection in their 1996 paper (J Appl. Cryst. 29, 584-587). Since 50% glycerol is kind of harsh, I wonder whether anyone has tried alternative cryo protectant. Your kind help will be highly appreciated. Another good reference: http://journals.iucr.org/j/issues/2002/05/00/do0015/index.html It suggests 35% PEG 400, 30% ethylene glycol, or 30% of whatever PG means (based on the rest of the paper I suspect propanediol, but the abbreviation doesn't really make sense - perhaps Eddie Snell can clarify). There are of course many other good cryoprotectants beyond those evaluated in the paper; personally, I'm a big fan of xylitol (which I believe will work in lower concentrations - at least with some conditions), but what really matters is what the crystals can tolerate. Note that these estimates are using very strict criteria - you can often get away with less cryoprotection if you are very good at freezing crystals and/or willing to tolerate some increased background. But I wouldn't try this until you've determined that your crystals can't handle the recommended amounts. -Nat
[ccp4bb] in vitro Tyr phosphorylation
Does anyone know of a good way to phosphorylate the Tyr on a protein for structural studies? Is there a generic kinase that can be coexpressed or purified for phosphorylation? The pCMF Amber codon system is very expensive and Glu really doesn't mimic pTyr all that well. Any ideas/help would be appreciated, G
[ccp4bb] How to config CPU to run ccp4 with multi-processor
Dear all I just installed CCP4 6.4 on Ubuntu 12.1. The program can work. I found it just used 1 processor instead of 4( Intel i5) for data solving. Does anyone know how to config CPU so that all processors can be used. Thanks Chang
[ccp4bb] How to config CPU to run ccp4 with multi-processor
Dear all I just installed CCP4 6.4 on Ubuntu 12.1. The program can work. I found it just used 1 processor instead of 4( Intel i5) for data solving. Does anyone know how to config CPU so that all processors can be used. Thanks Chang
