[ccp4bb] mtz for zanuda - which symmetry?
Hi - For Zanuda, the mtz input it wants: what spacegroup are the data meant to be merged to? The highest possible, potentially twinned? Or the lowest possible? I've looked reasonably hard (10min on the manuals and The Google), and the answer did not leap out at me at all. Even though it seems a rather crucial question - or is it not, in which case, why not, what am I missing? phx
[ccp4bb] השב: Re: [ccp4bb] Curious electron density associated with Asp sidechain
Hi, it looks like gly-asp not asp-gly in this case, doesn'it? Cheers, Boaz הודעה מקורית מאת: Jonathan Cooper bogba...@yahoo.co.uk תאריך: אל: CCP4BB@JISCMAIL.AC.UK נושא: Re: [ccp4bb] Curious electron density associated with Asp sidechain Hello Tony is that Asp-Gly? If so, it could be prone to succinimide formation. Check out this paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323960/ and references therein! Good luck Jon.Cooper --- On Thu, 25/4/13, Antony Oliver antony.oli...@sussex.ac.uk wrote: From: Antony Oliver antony.oli...@sussex.ac.uk Subject: [ccp4bb] Curious electron density associated with Asp sidechain To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, 25 April, 2013, 17:10 Dear CCP4 colleagues. I'm just finishing up a refinement, but am left with one little curio that I just can't seem to solve. One aspartic acid residue is associated with some extra, unexplained electron density. -- please see: http://i.imgur.com/vCYOqam.png Where, the Fo-Fc map is contoured at 3.78 rsmd in Coot. I have tried a number of different modelling scenarios, but as yet can't reach a wholly satisfactory conclusion; waters, alternate conformers, really don't seem to cut it. I though about some radiation-induced phenomena, but this data set was collected on a home-source, so I guess this is unlikely. So, I would really appreciate some ideas and suggestions. Hopefully it is blindingly obvious to someone. Random Thought: could it be PEGylation of the side-chain? Some other hopefully useful background information: * I'm sure it is/was an ASP, because the same protein (made from the same construct) has been used in previous crystallisations, and the resultant structures have clear, unambiguous electron density for the side chain. * the crystallization condition is PEG 200, with some Na/K phosphate at pH 5.8, and NaCl. The protein itself contains HEPES buffer. With many thanks, Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.uk tel (office): 44 (0)1273 678349 tel (lab): 44 (0)1273 677512
Re: [ccp4bb] השב: Re: [ccp4bb] Curious electron density associated with Asp sidechain
could this also be an alternative C-Terminus at lower occupancy? Regards Kornelius On Fri, Apr 26, 2013 at 10:45 AM, Boaz Shaanan bshaa...@exchange.bgu.ac.ilwrote: Hi, it looks like gly-asp not asp-gly in this case, doesn'it? Cheers, Boaz הודעה מקורית מאת: Jonathan Cooper bogba...@yahoo.co.uk תאריך: אל: CCP4BB@JISCMAIL.AC.UK נושא: Re: [ccp4bb] Curious electron density associated with Asp sidechain Hello Tony is that Asp-Gly? If so, it could be prone to succinimide formation. Check out this paper: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2323960/ and references therein! Good luck Jon.Cooper --- On *Thu, 25/4/13, Antony Oliver antony.oli...@sussex.ac.uk* wrote: From: Antony Oliver antony.oli...@sussex.ac.uk Subject: [ccp4bb] Curious electron density associated with Asp sidechain To: CCP4BB@JISCMAIL.AC.UK Date: Thursday, 25 April, 2013, 17:10 Dear CCP4 colleagues. I'm just finishing up a refinement, but am left with one little curio that I just can't seem to solve. One aspartic acid residue is associated with some extra, unexplained electron density. --please see: http://i.imgur.com/vCYOqam.png Where, the Fo-Fc map is contoured at 3.78 rsmd in Coot. I have tried a number of different modelling scenarios, but as yet can't reach a wholly satisfactory conclusion; waters, alternate conformers, really don't seem to cut it. I though about some radiation-induced phenomena, but this data set was collected on a home-source, so I guess this is unlikely. So, I would really appreciate some ideas and suggestions. Hopefully it is blindingly obvious to someone. Random Thought: could it be PEGylation of the side-chain? Some other hopefully useful background information: * I'm sure it is/was an ASP, because the same protein (made from the same construct) has been used in previous crystallisations, and the resultant structures have clear, unambiguous electron density for the side chain. * the crystallization condition is PEG 200, with some Na/K phosphate at pH 5.8, and NaCl. The protein itself contains HEPES buffer. With many thanks, Tony. --- Dr Antony W Oliver Senior Research Fellow CR-UK DNA Repair Enzymes Group Genome Damage and Stability Centre Science Park Road University of Sussex Falmer, Brighton, BN1 9RQ email: antony.oli...@sussex.ac.ukhttp://mc/compose?to=antony.oli...@sussex.ac.uk tel (office): +44 (0)1273 678349 tel (lab): +44 (0)1273 677512 -- *Kornelius Zeth* *Email: kornelius.z...@gmail.com* *Unidad de Biofisica (CSIC-UPV/EHU) Barrio Sarriena s/n 48940, Leioa, Vizcaya* *SPAIN*
[ccp4bb] Factor 10a
Dear colleagues, i hope every one doing well. i am trying to remove the 10x his tag , as i tried to crystallize with the tag but i didn^t obtain crystals unless some salt crystals , very small crystals and some precipitant. i want to use factor 10a protease but i didn^t tried it before. this is the first time for using it. i have factor 10a protease from Qiagen 400 unit . i have read the protocol but i still need some suggestion and experience as you know it is expensive. also i have another question , how many protein digested by one unit ? i know it depend but roughly how much? i really appreciate your support. best regards Amr
Re: [ccp4bb] mtz for zanuda - which symmetry?
Hi Frank, I have not worked with Zanuda, but googled for the manual (first hit; 1sec). It seems that there are 2 modes: 1) to restore the true (higher symmetry) space group after a structure had intentionally been solved in a lower symmetry space group e.g. P1. In this case the option to merge to the highest possible symmetry space group is what would come to my mind. 2) to look for pseudosymmetry. Since pseudosymmetry might have been interpreted as crystallographic symmetry during the previous attempts to process the data, here the option to merge to the lowest possible symmetry would seem most adequate. If there would be reasons to suspect that the data might be potentially twinned, there seems to be a third option available. Of course, there might be a combination of twinning and pseudosymmetry, intentionally attempted to be solved at lower symmetry... For hard problems (in my experience usually the case) one has to try everything. Good luck! herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft Sent: Friday, April 26, 2013 10:18 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] mtz for zanuda - which symmetry? Hi - For Zanuda, the mtz input it wants: what spacegroup are the data meant to be merged to? The highest possible, potentially twinned? Or the lowest possible? I've looked reasonably hard (10min on the manuals and The Google), and the answer did not leap out at me at all. Even though it seems a rather crucial question - or is it not, in which case, why not, what am I missing? phx
Re: [ccp4bb] mtz for zanuda - which symmetry?
Yes I know all that; but where are the explicit instructions? (I assume they're there, I just couldn't see them.) On 26/04/2013 10:27, herman.schreu...@sanofi.com wrote: Hi Frank, I have not worked with Zanuda, but googled for the manual (first hit; 1sec). It seems that there are 2 modes: 1) to restore the true (higher symmetry) space group after a structure had intentionally been solved in a lower symmetry space group e.g. P1. In this case the option to merge to the highest possible symmetry space group is what would come to my mind. 2) to look for pseudosymmetry. Since pseudosymmetry might have been interpreted as crystallographic symmetry during the previous attempts to process the data, here the option to merge to the lowest possible symmetry would seem most adequate. If there would be reasons to suspect that the data might be potentially twinned, there seems to be a third option available. Of course, there might be a combination of twinning and pseudosymmetry, intentionally attempted to be solved at lower symmetry... For hard problems (in my experience usually the case) one has to try everything. Good luck! herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft Sent: Friday, April 26, 2013 10:18 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] mtz for zanuda - which symmetry? Hi - For Zanuda, the mtz input it wants: what spacegroup are the data meant to be merged to? The highest possible, potentially twinned? Or the lowest possible? I've looked reasonably hard (10min on the manuals and The Google), and the answer did not leap out at me at all. Even though it seems a rather crucial question - or is it not, in which case, why not, what am I missing? phx
Re: [ccp4bb] mtz for zanuda - which symmetry?
Input pdb and mtz should form a valid input for refmac, hence the same cell and sym and merged mtz. On 26 Apr 2013, at 09:17, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Hi - For Zanuda, the mtz input it wants: what spacegroup are the data meant to be merged to? The highest possible, potentially twinned? Or the lowest possible? I've looked reasonably hard (10min on the manuals and The Google), and the answer did not leap out at me at all. Even though it seems a rather crucial question - or is it not, in which case, why not, what am I missing? phx -- Scanned by iCritical.
Re: [ccp4bb] LINK or LINKR
On 04/26/2013 12:29 PM, Eleanor Dodson wrote: Is there any consensus about the accepted format for this? I believe Garib uses LINKR to add a link name to the record, (cant find a description in the documentation though???) but also in the documentation REFMAC is said to provide a link between symmetry related like this with the target distance here LINK1P DG A 11.61000 O3' DC A 2 1555 6554 i But REFMAC a) ignores the given distance and b) writes it out as : LINK PDG A 1 O3' DC A 2 1555 2554 1.48 This is in agreement with the PDB definition but with a wrong distance - presumably derived in the innards of the dictionary: PDB LINK definition 12345678901234567890123456789012345678901234567890123456789012345678901234567890 LINK O GLY A 49NANA A6001 1555 1555 2.98 LINK OG1 THR A 51NANA A6001 1555 1555 2.72 12345678901234567890123456789012345678901234567890123456789012345678901234567890 LINK O GLY A 49NANA A6001 1555 1555 2.98 LINK OG1 THR A 51NANA A6001 1555 1555 2.72 coot seems to refuse to read the LINKR at all! As for the last bit, do you mean 1) that coot gives a parsing error when reading files with LINKRs or 2) that coot otherwise parses such files OK, but skips past LINKR records (and in that case, what would you have it do?) Which version of mmdb is the coot that you are using linked against? Paul.
Re: [ccp4bb] LINK or LINKR
Hi Eleanor, The recent versions of Refmac work well with the records in PDB format. According to the list of bug fixes on the website, Refmac should now take the distance from the PDB file (it used to complain about the distance record). Changing the 1.48 to 1.61 in the new LINK record should do the trick. So far for the theory, in practice there are still a lot of difficulties dealing with LINKs. 1) I noticed the LINK record in the output has a different symmetry record, are the two equivalent? 2) The PDB generates LINK records upon deposition, even for things that were not restrained by LINKs in Refinement, which may misrepresent the refinement. 3) The LINK records in the PDB give the actual distance, not the target. Which means that you can accidentally replace good restraint targets with poor ones, simply by loading a (previously poorly refined or miss-annotated) PDB file. 4) There is no consensus dictionary or a repository for LINKs at the PDB. The CCP4 dictionary has a number of LINKs, but is quite incomplete. 5) Some target LINK lengths, especially in ion coordination, vary with context even if the involved atoms are the same. Cheers, Robbie From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor Dodson Sent: Friday, April 26, 2013 13:30 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] LINK or LINKR Is there any consensus about the accepted format for this? I believe Garib uses LINKR to add a link name to the record, (cant find a description in the documentation though???) but also in the documentation REFMAC is said to provide a link between symmetry related like this with the target distance here LINK1P DG A 11.61000 O3' DC A 2 1555 6554 i But REFMAC a) ignores the given distance and b) writes it out as : LINK PDG A 1 O3' DC A 2 1555 2554 1.48 This is in agreement with the PDB definition but with a wrong distance - presumably derived in the innards of the dictionary: PDB LINK definition 1234567890123456789012345678901234567890123456789012345678901234567890123456 7890 LINK O GLY A 49NANA A6001 1555 1555 2.98 LINK OG1 THR A 51NANA A6001 1555 1555 2.72 1234567890123456789012345678901234567890123456789012345678901234567890123456 7890 LINK O GLY A 49NANA A6001 1555 1555 2.98 LINK OG1 THR A 51NANA A6001 1555 1555 2.72 coot seems to refuse to read the LINKR at all! Confused Eleanor
Re: [ccp4bb] LINK or LINKR
First for Paul and Eugene: I am using the version of coot labelled in brown coot - 0.7 Clayton COOT_PREFIX is /y/prog/linux/xtal/coot/coot-Linux-i386-fedora-12-gtk2-python /y/programs/xtal/coot/coot-Linux-i386-fedora-12-gtk2-python//bin/coot-real Acquiring application resources from /y/prog/linux/xtal/coot/coot-Linux-i386-fedora-12-gtk2-python/share/coot/cootrc INFO:: splash_screen_pixmap_dir /y/prog/linux/xtal/coot/coot-Linux-i386-fedora-12-gtk2-python/share/coot/pixmaps INFO:: Colours file: /y/prog/linux/xtal/coot/coot-Linux-i386-fedora-12-gtk2-python/share/coot/colours.def loaded INFO:: Using Standard CCP4 Refmac dictionary from CLIBD_MON: /y/programs/xtal/ccp4-6.3.0/lib/data/monomers/ There are 125 data in /y/programs/xtal/ccp4-6.3.0/lib/data/monomers//list/mon_lib_list.cif If it tries to read a LINKR record, it just bombs out saying no spacegroup given! Presumably it slips over into the CRYST1 record trying to find something (maybe the LINK name?) and thus ignores the CRYST1 information? And thank you to Robbie for that clarification - who should be recruited to try and make more sense of the situation? Eleanor On 26 April 2013 13:03, Robbie Joosten robbie_joos...@hotmail.com wrote: Hi Eleanor, ** ** The recent versions of Refmac work well with the records in PDB format. According to the list of bug fixes on the website, Refmac should now take the distance from the PDB file (it used to complain about the distance record). Changing the 1.48 to 1.61 in the new LINK record should do the trick. So far for the theory, in practice there are still a lot of difficulties dealing with LINKs. ** ** **1) **I noticed the LINK record in the output has a different symmetry record, are the two equivalent? **2) **The PDB generates LINK records upon deposition, even for things that were not restrained by LINKs in Refinement, which may misrepresent the refinement. **3) **The LINK records in the PDB give the actual distance, not the target. Which means that you can accidentally replace good restraint targets with poor ones, simply by loading a (previously poorly refined or miss-annotated) PDB file. **4) **There is no consensus dictionary or a repository for LINKs at the PDB. The CCP4 dictionary has a number of LINKs, but is quite incomplete. **5) **Some target LINK lengths, especially in ion coordination, vary with context even if the involved atoms are the same. ** ** Cheers, Robbie ** ** ** ** *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of *Eleanor Dodson *Sent:* Friday, April 26, 2013 13:30 *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* [ccp4bb] LINK or LINKR ** ** Is there any consensus about the accepted format for this? I believe Garib uses LINKR to add a link name to the record, (cant find a description in the documentation though???) but also in the documentation REFMAC is said to provide a link between symmetry related like this with the target distance here LINK1P DG A 11.61000 O3' DC A 2 1555 6554 i But REFMAC a) ignores the given distance and b) writes it out as : LINK PDG A 1 O3' DC A 2 1555 2554 1.48 This is in agreement with the PDB definition but with a wrong distance - presumably derived in the innards of the dictionary: ** ** PDB LINK definition 12345678901234567890123456789012345678901234567890123456789012345678901234567890 LINK O GLY A 49NANA A6001 1555 1555 2.98 LINK OG1 THR A 51NANA A6001 1555 1555 2.72 12345678901234567890123456789012345678901234567890123456789012345678901234567890 LINK O GLY A 49NANA A6001 1555 1555 2.98 LINK OG1 THR A 51NANA A6001 1555 1555 2.72 ** ** coot seems to refuse to read the LINKR at all! Confused Eleanor
Re: [ccp4bb] Anomalous atom or ligand?
Not sure I follow you completely.. You dont say what anom scatterer you expect, or what the wavelength is. 1) How did you find the anomalous peaks? If from an anomalous difference Fourier there will inevitably be noise. In practice when there are S to check this can be very helpful in deciding on noise levels. You can be pretty sure that SG and SD are indeed S, and if the wave length is appropriate, that they should show some anom. signal. One often finds the well defined S show up well. then there is a grey area wher they are down amongst noise but still appear. It often highlights how many MET are in alternate conformations.. If possible I like to use these as bench marks for P. But in my experience the P are often even weaker - maybe the ligand is not fully occupied, or the P temp. factors are higher? On 24 April 2013 10:56, Kavyashree Manjunath ka...@ssl.serc.iisc.in wrote: Dear users, After detecting the anomalous peaks in a data, Is it necessary that there will be an anomalous atom in most of the peaks? In a particular case, a low ranking peak was assigned an anomalous atom because it was present in the native structure, while a peak with a rank higher than this one did not correspond to anomalous position in native structure. For eg. Peak 15 in ligand bound structure corresponds to the anomalous position in native structure, so anomalous atom was assigned. But Peak 3,4 in ligand bound structure does not correspond to anomalous position in native structure but it is present near the ligand which is beta and gamma Phosphates of ATP. The question is whether It is ATP or AMP and 2 anomalous atoms? Thanking you Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
[ccp4bb] Workshop focused on exploitation of Synchrotron Radiation Circular Dichroism for Membrane Protein and Carbohydrate Research - 31 May 2013, Leeds
Dear all, those of you working in the membrane protein and/or carbohydrate field may be very interested in attending a 1 day workshop which will highlight the unique capabilities of the CD beamline B23, at Diamond. The workshop is focused on how the technique can aid your research in these key areas. The workshop will be a dynamic mix of introducing beamline basics, how to get beamtime, sample requirements experimental possibilities as well as a series of science case studies from recent users of the beamline and ample time for discussions with both beamline staff and experienced users. There is a limited number of places so register now to avoid disappointment! Full details and registration for the workshop are available from the this link: Diamond B23 circular dichroism beamline Workshop Series / University of Leeds / University of Liverpoolhttp://www.diamond.ac.uk/Home/Events/B23-Workshop-Series.html B23 Roadshow Series - Membrane Protein and Carbohydrate Research Workshop Friday 31 May 2013, The Metropole Hotel, Leeds Hosted by: Diamond Light Source, University of Leeds and University of Liverpool On the 31th May 2013, Diamond Light Source (DLS), together with the University of Leeds and University of Liverpool will hold a one-day workshop dedicated to membrane protein and carbohydrate research, with a particular view to promote the synchrotron based CD beamline B23 for these biological materials. The event will be held at The Metropole Hotelhttp://www.metropole-hotel.co.uk/, Leeds, a convenient location well accessible using public transport. The workshop is free to attend and includes refreshments and lunch, but registration is limited to 50 places. Successful registrants will be expected to reconfirm their place nearer the time in order to give those on a reserved list a chance to take up any cancellation places. Confirmed speakers Simon Patching, Leeds University Konstantinos Beis, Imperial College London Bonnie Wallace, Birkbeck College Marcelo Lima, Federal University of Sao Paolo David Middleton, Liverpool University Paul Walton, York University Alex Cameron, Warwick University Giuliano Siligardi Rohanah Hussain, Diamond Light Source B23 For details on the workshop and to register, please visit the websitehttp://www.diamond.ac.uk/Home/Events/B23-Workshop-Series.html. This will be the second event in a series of Road shows to highlight Diamond Light Source B23 throughout the country. The next event is scheduled to take place in Liverpool on the 1st November 2013 on the topic of Material Science. If you are interested in participating in future events please email Rohanah Hussainmailto:rohanah.huss...@diamond.ac.uk. With best wishes, Rohanah Hussain, Giuliano Siligardi, Peter Henderson and Edwin Yates.
[ccp4bb] Postdoctoral position in structural virology at the Scripps Research Institute
A postdoctoral position is available starting immediately to continue the structural work on adenovirus capsids and associated proteins (Reddy et al., Crystal structure of human adenovirus at 3.5Å resolution. Science. 2010;329(5995):1071-5) at The Scripps Research Institute, La Jolla, California, in the research group of Vijay Reddy. Candidates with a Ph.D in biochemistry or structural biology and within 2-3 years of their graduation are encouraged to apply. Hands on experience and knowledge in protein expression, purification and aspects of structure determination employing x-ray crystallographic methods are essential for the intended position. Interested candidates are requested to send their CV, summary of research interests and names of 3 references to red...@scripps.edu Thank you Vijay S. Reddy, Ph.D. Associate Professor Integrative Structural and Computational Biology, TPC6 The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037 E-mail: red...@scripps.edu WWW:http://www.scripps.edu/~reddyv VIPERdb: http://viperdb.scripps.edu Phone: (858) 784-8191 FAX: (858) 784-8896
[ccp4bb] Off-topic: Carboxypeptidase
Hi All: I wish to identify and remove unstructured regions at the C-terminus of my protein for crystallization and am considering using carboxypeptidase digestions to achieve this. 1) I would like a protocol and your preferred source of commercial carboxypeptidases as well their storage conditions. 2) Do I need to use a mixture of different carboxypeptidases? If so, which ones? 3) Is ESI-MS intact mass analysis a good way to identify the post-treatment C-terminus or does this treatment typically yield a large mixture of C-termini? 4) Any published references to this approach would be appreciated. Thank you. Best, Vish
[ccp4bb] refinement hanging--what am I missing?
Hi all, Here is a problem that's been annoying me, and demanding levels of thought all out of proportion with the importance of the project: I have two related crystal forms of the same small protein. In both cases, the data look quite decent, and extend beyond 2 A, but the refinement stalls with statistics that are just bad enough to make me deeply uncomfortable. However, the maps look pretty good, and there's no obvious path to push the refinement further. Xtriage doesn't raise any red flags, nor does running the data through the Yeates twinning server. Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution of data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27 Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26 As you would expect, the packing is essentially the same in both crystal forms. It's interesting to note (but is it relevant?) that the packing is quite dense--solvent content is only 25-30%. This kind of stalling at high R values smells like a twin problem, but it's not clear to me what specific kind of twinning might explain this behavior. Any thoughts about what I might be missing here? Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] refinement hanging--what am I missing?
Pat, Try TLS - I usually don't invoke it at this type of resolution but in one case I saw it make a surprisingly significant improvement. I would also be tempted to put the structures through Arp/wArp and see if it lowers the R-free any more - rightly or wrongly I view this as the lowest reasonably achievable R-factor with isotropic modeling - and especially look at the maps after it has finished in case it shows up anything you had missed. When I had P21 - P2x212x twinning the R-free held up in the mid-30's at 2 Angstrom resolution so absent any indications in Truncate or Xtriage I wouldn't suggest that. A final question is how much disordered structure is missing from your models ? Could a partly ordered but unmodeled segment be driving up R-free ? Cheers Phil Jeffrey Princeton On 4/26/13 5:38 PM, Patrick Loll wrote: Hi all, Here is a problem that's been annoying me, and demanding levels of thought all out of proportion with the importance of the project: I have two related crystal forms of the same small protein. In both cases, the data look quite decent, and extend beyond 2 A, but the refinement stalls with statistics that are just bad enough to make me deeply uncomfortable. However, the maps look pretty good, and there's no obvious path to push the refinement further. Xtriage doesn't raise any red flags, nor does running the data through the Yeates twinning server. Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution of data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27 Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26 As you would expect, the packing is essentially the same in both crystal forms. It's interesting to note (but is it relevant?) that the packing is quite dense--solvent content is only 25-30%. This kind of stalling at high R values smells like a twin problem, but it's not clear to me what specific kind of twinning might explain this behavior. Any thoughts about what I might be missing here? Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] refinement hanging--what am I missing?
Responding to a couple of questions from Ethan, Charlie, and Phil: Ethan: Using the default bulk solvent modeling in Phenix; no selenium, but I'll double-check wavelengths as a sanity check for scattering factors (but several other native data sets from the same synchrotron trip refined beautifully, so I suspect there's no gross boo-boos of this nature...) Charlie: Solvent regions are pretty clean; I haven't tried any flipping (these are molecular replacement models, so it didn't occur to me...). I tried applying NCS in one case (the smaller cell) and it had no apparent effect on the refinement. The Fo-Fc map has no strong features crying out for interpretation. Just based on geometry and map appearance, I'd be inclined to say the refinement is done, were it not for the crappy R values. Phil: I used TLS for refinement in both xtal forms; it gives a small improvement (0.01-0.03) in Rfree in both cases, but nothing magic. I simply used one monomer/TLS group (these are ubiquitin variants, so the monomer itself is pretty much a little rock, without any internal domain motions). There are the usual complement of disordered side chains, but nothing unusual, and 98% of the main chain is accounted for. Haven't tried Arp/wArp yet... Excellent thoughts, keep those cards and letters coming. I'm still chewing on the substantive comments from Dean and Adrian... Thanks, Pat On 26 Apr 2013, at 6:17 PM, Carter, Charlie wrote: Hi Pat, Your stats aren't all that bad, but I share your discomfort. Do the solvent regions retain any significant features? Have you tried flipping those features? Have you applied NCS? What does the Fo - Fc map look like? Charlie On Apr 26, 2013, at 5:38 PM, Patrick Loll wrote: Hi all, Here is a problem that's been annoying me, and demanding levels of thought all out of proportion with the importance of the project: I have two related crystal forms of the same small protein. In both cases, the data look quite decent, and extend beyond 2 A, but the refinement stalls with statistics that are just bad enough to make me deeply uncomfortable. However, the maps look pretty good, and there's no obvious path to push the refinement further. Xtriage doesn't raise any red flags, nor does running the data through the Yeates twinning server. Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution of data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27 Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26 As you would expect, the packing is essentially the same in both crystal forms. It's interesting to note (but is it relevant?) that the packing is quite dense--solvent content is only 25-30%. This kind of stalling at high R values smells like a twin problem, but it's not clear to me what specific kind of twinning might explain this behavior. Any thoughts about what I might be missing here? Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] refinement hanging--what am I missing?
Hi Pat, On top of Phil's suggestion for invoking TLS, what about the NCS? Is there anything peculiar about them (tNCS or the like)? If there isn't, are you imposing NCS during refinement? perhaps you should relax the NCS restraints or not impose any at all and see what happens then? My 2p thoughts. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Patrick Loll [pat.l...@drexel.edu] Sent: Saturday, April 27, 2013 12:38 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] refinement hanging--what am I missing? Hi all, Here is a problem that's been annoying me, and demanding levels of thought all out of proportion with the importance of the project: I have two related crystal forms of the same small protein. In both cases, the data look quite decent, and extend beyond 2 A, but the refinement stalls with statistics that are just bad enough to make me deeply uncomfortable. However, the maps look pretty good, and there's no obvious path to push the refinement further. Xtriage doesn't raise any red flags, nor does running the data through the Yeates twinning server. Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution of data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27 Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26 As you would expect, the packing is essentially the same in both crystal forms. It's interesting to note (but is it relevant?) that the packing is quite dense--solvent content is only 25-30%. This kind of stalling at high R values smells like a twin problem, but it's not clear to me what specific kind of twinning might explain this behavior. Any thoughts about what I might be missing here? Thanks, Pat --- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
Re: [ccp4bb] refinement hanging--what am I missing?
Hi Patrick, Did you try using a different refinement program (e.g. Refmac)? Which type of NCS restraints did you use, global or local (torsion- or distance-based)? Have you tried optimizing your restraint weights? Have you tried running a huge number of refinement cycles? You can also try running PDB_REDO (plug plug) which will try a number of things to improve your model. Cheers, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Loll Sent: Saturday, April 27, 2013 00:32 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] refinement hanging--what am I missing? Responding to a couple of questions from Ethan, Charlie, and Phil: Ethan: Using the default bulk solvent modeling in Phenix; no selenium, but I'll double-check wavelengths as a sanity check for scattering factors (but several other native data sets from the same synchrotron trip refined beautifully, so I suspect there's no gross boo-boos of this nature...) Charlie: Solvent regions are pretty clean; I haven't tried any flipping (these are molecular replacement models, so it didn't occur to me...). I tried applying NCS in one case (the smaller cell) and it had no apparent effect on the refinement. The Fo-Fc map has no strong features crying out for interpretation. Just based on geometry and map appearance, I'd be inclined to say the refinement is done, were it not for the crappy R values. Phil: I used TLS for refinement in both xtal forms; it gives a small improvement (0.01-0.03) in Rfree in both cases, but nothing magic. I simply used one monomer/TLS group (these are ubiquitin variants, so the monomer itself is pretty much a little rock, without any internal domain motions). There are the usual complement of disordered side chains, but nothing unusual, and 98% of the main chain is accounted for. Haven't tried Arp/wArp yet... Excellent thoughts, keep those cards and letters coming. I'm still chewing on the substantive comments from Dean and Adrian... Thanks, Pat On 26 Apr 2013, at 6:17 PM, Carter, Charlie wrote: Hi Pat, Your stats aren't all that bad, but I share your discomfort. Do the solvent regions retain any significant features? Have you tried flipping those features? Have you applied NCS? What does the Fo - Fc map look like? Charlie On Apr 26, 2013, at 5:38 PM, Patrick Loll wrote: Hi all, Here is a problem that's been annoying me, and demanding levels of thought all out of proportion with the importance of the project: I have two related crystal forms of the same small protein. In both cases, the data look quite decent, and extend beyond 2 A, but the refinement stalls with statistics that are just bad enough to make me deeply uncomfortable. However, the maps look pretty good, and there's no obvious path to push the refinement further. Xtriage doesn't raise any red flags, nor does running the data through the Yeates twinning server. Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution of data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27 Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26 As you would expect, the packing is essentially the same in both crystal forms. It's interesting to note (but is it relevant?) that the packing is quite dense- -solvent content is only 25-30%. This kind of stalling at high R values smells like a twin problem, but it's not clear to me what specific kind of twinning might explain this behavior. Any thoughts about what I might be missing here? Thanks, Pat - -- Patrick J. Loll, Ph. D. Professor of Biochemistry Molecular Biology Director, Biochemistry Graduate Program Drexel University College of Medicine Room 10-102 New College Building 245 N. 15th St., Mailstop 497 Philadelphia, PA 19102-1192 USA (215) 762-7706 pat.l...@drexelmed.edu
[ccp4bb] STRAP secondary structure
Hello, Every one, I am preparing a ppt presentation in an emergency style. What I want is a graph of sequence alignment with secondary structure on the top. Using STRAP i can got alignment nicely, but did not know the tips to add secondary structure on top. Anybody of experienced can give me a hand? Tutorial-like instruction would be much helpful. Your kindness is greatly appreciated. Thanks Mike
Re: [ccp4bb] Anomalous atom or ligand?
Respected Mam, I used Cadmium at home source 1.54179Ang. For detecting the anomalous peaks CAD and FFT was used. It has Se also. So while refining the occupancies of Cd, Se, S and P are also refined. Thanking you Respectfully Kavya Not sure I follow you completely.. You dont say what anom scatterer you expect, or what the wavelength is. 1) How did you find the anomalous peaks? If from an anomalous difference Fourier there will inevitably be noise. In practice when there are S to check this can be very helpful in deciding on noise levels. You can be pretty sure that SG and SD are indeed S, and if the wave length is appropriate, that they should show some anom. signal. One often finds the well defined S show up well. then there is a grey area wher they are down amongst noise but still appear. It often highlights how many MET are in alternate conformations.. If possible I like to use these as bench marks for P. But in my experience the P are often even weaker - maybe the ligand is not fully occupied, or the P temp. factors are higher? On 24 April 2013 10:56, Kavyashree Manjunath ka...@ssl.serc.iisc.in wrote: Dear users, After detecting the anomalous peaks in a data, Is it necessary that there will be an anomalous atom in most of the peaks? In a particular case, a low ranking peak was assigned an anomalous atom because it was present in the native structure, while a peak with a rank higher than this one did not correspond to anomalous position in native structure. For eg. Peak 15 in ligand bound structure corresponds to the anomalous position in native structure, so anomalous atom was assigned. But Peak 3,4 in ligand bound structure does not correspond to anomalous position in native structure but it is present near the ligand which is beta and gamma Phosphates of ATP. The question is whether It is ATP or AMP and 2 anomalous atoms? Thanking you Regards Kavya -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean.
