Re: [ccp4bb] refinement hanging--what am I missing?
Dear Patrick, I don't know how many comments you got offline, but here are my comments: 1) Ice rings. Even rings that are hardly visible by eye can disturb the data such that Rfactors get stuck in the range you mention. Especially XDS, which is otherwise an excellent data processing program, performs poor in the presence of ice rings. You may want to look carefulla at your diffraction images, the wilson plot and at which resolutions you find most outlyers. 2) Crystal packing. With 2 or 4 molecules in the asymmetric unit, a~b or 2a~b and space group P(2)(1)2(1)(2)(1), you have plenty possibilities for pseudo crystallographic symmetry. Since you have high resolution data, I would process or expand the data to P1, also expand your pdb file to P1 and refine in P1 to see what happens. I would also run Phaser or some other molecular replacement program on the P1 data to see what comes out. Good luck! Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Patrick Loll Sent: Saturday, April 27, 2013 12:32 AM 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
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
There isnt much information here! Funny that 3 chains are poor - does that mean one and a half heterodimers? I presume you have checked spacegroup? Zanuda will test to see if any higher symmetry is present.. Eleanor On 29 April 2013 06:02, sonali dhindwal sonali11dhind...@yahoo.co.inwrote: Dear All, We are working on a crystal structure with 12 molecules in an asymmetric unit. It is a heteromer and each chain is made up of two type of chains. Therefore there are 24 chains in an asymmetric unit. It is giving solution in P1. Rfactor and Rfree has reached 20.25 and 25.6 at a resolution of 1.8Angstrom All the chains are refining well and have good electron density except 3 chains which have very poor electron density in most of the region. Whereas, the crystal structure of variants of the same protein we have solved earlier refined well and has very good electron density in all the chains. What could be the possible reason for these three chains which are not refining properly. Any suggestions will be very useful. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.”
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
Dear Eleanor, Thanks for the suggestion, I just checked on the Zanuda program, it is also giving P1 as the best possible spacegroup for the molecule. and by not refining well, I meant for the electron density which is broken at many places at main chain, and poor electron density for the side chains which are otherwise good in other chains. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” From: Eleanor Dodson eleanor.dod...@york.ac.uk To: CCP4BB@JISCMAIL.AC.UK Sent: Monday, 29 April 2013 4:36 PM Subject: Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit There isnt much information here! Funny that 3 chains are poor - does that mean one and a half heterodimers? I presume you have checked spacegroup? Zanuda will test to see if any higher symmetry is present.. Eleanor On 29 April 2013 06:02, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: Dear All, We are working on a crystal structure with 12 molecules in an asymmetric unit. It is a heteromer and each chain is made up of two type of chains. Therefore there are 24 chains in an asymmetric unit. It is giving solution in P1. Rfactor and Rfree has reached 20.25 and 25.6 at a resolution of 1.8Angstrom All the chains are refining well and have good electron density except 3 chains which have very poor electron density in most of the region. Whereas, the crystal structure of variants of the same protein we have solved earlier refined well and has very good electron density in all the chains. What could be the possible reason for these three chains which are not refining properly. Any suggestions will be very useful. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.”
Re: [ccp4bb] refinement hanging--what am I missing?
herman.schreu...@sanofi.com wrote: I would process or expand the data to P1, also expand your pdb file to P1 and refine in P1 to see what happens. I would also run Phaser or some other molecular replacement program on the P1 data to see what comes out. process or expand? I don't understand how there can really be a choice here. If the data is expanded from higher symmetry, it will precisely have that higher symmetry, i.e. all reflections that were equivalent in the higher symmetry will be identical. Any information about asymmetry was lost when the data were merged in the higher symmetry space group. Won't refinement by minimizing a target function have exactly the same solution? Or not exactly, because the refinement program can introduce asymmetry by allowing different phases for the equivalent reflections. But where is the information to generate that asymmetry coming from? There is probably something I'm missing here, but i would definitely reprocess in the lower symmetry if the rotation angle was sufficient to give good completeness. eab
Re: [ccp4bb] refinement hanging--what am I missing?
Hi Edward, I and also others in the bulletin boards have had cases where the protein would build e.g. tetramers with internal 222 symmetry, which would pack with the 2-folds parallel to the crystallographic 2-folds with say one 2-fold a little shifted from the crystallographic position. In these cases, the internal 222 symmetry overwhelmed the contribution of the small shift and all processing programs, including Pointless would insist that the crystal was P2x2x2x. Also the Rfactors (Rsym, Rmeas) would be the same whether processing in P222 or P2. However, the only way to pack the proteins correctly in the unit cell, would be to process in P2 or even in P1 and run molecular replacement in P2 or P1 and than reconstruct the most likely true space group, for which now the program Zanuda exists. Since in these cases the diffraction patterns would have perfect (within experimental error) P222 symmetry, I think it would be valid to expand the P222 data to P2 of P1. Of course collecting the full data set is still the best way to go. The asymmetry is generated by the way the molecular replacement program places the molecules in the asymmetric unit and will, as you mention, only be in the phases. Expanding to P1 and running molecular replacement in P1 would test all possibilities at once, but the signal would be weaker. A more cautious approach would be to process in the three possible P2 spacegroups (choosing the 2-fold along a, b or c) and run molecular replacement for each option. It may also be that your problem lies elsewhere. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. Berry Sent: Monday, April 29, 2013 4:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] refinement hanging--what am I missing? herman.schreu...@sanofi.com wrote: I would process or expand the data to P1, also expand your pdb file to P1 and refine in P1 to see what happens. I would also run Phaser or some other molecular replacement program on the P1 data to see what comes out. process or expand? I don't understand how there can really be a choice here. If the data is expanded from higher symmetry, it will precisely have that higher symmetry, i.e. all reflections that were equivalent in the higher symmetry will be identical. Any information about asymmetry was lost when the data were merged in the higher symmetry space group. Won't refinement by minimizing a target function have exactly the same solution? Or not exactly, because the refinement program can introduce asymmetry by allowing different phases for the equivalent reflections. But where is the information to generate that asymmetry coming from? There is probably something I'm missing here, but i would definitely reprocess in the lower symmetry if the rotation angle was sufficient to give good completeness. eab
Re: [ccp4bb] refinement hanging--what am I missing?
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hi Ed, just FYI: while this does not apply in the ccp4/mtz world, the fact you might be missing is that in some refinement environments merging is carried out by the refinement program so that the actual data can be kept unmerged and e.g. the information you refer to does not get lost (although scaling would smoothen it to a certain extent but at least does not erase it as mergin does). In that case you could expand to P1 (as far as I understand). best, Tim On 04/29/2013 04:13 PM, Edward A. Berry wrote: herman.schreu...@sanofi.com wrote: I would process or expand the data to P1, also expand your pdb file to P1 and refine in P1 to see what happens. I would also run Phaser or some other molecular replacement program on the P1 data to see what comes out. process or expand? I don't understand how there can really be a choice here. If the data is expanded from higher symmetry, it will precisely have that higher symmetry, i.e. all reflections that were equivalent in the higher symmetry will be identical. Any information about asymmetry was lost when the data were merged in the higher symmetry space group. Won't refinement by minimizing a target function have exactly the same solution? Or not exactly, because the refinement program can introduce asymmetry by allowing different phases for the equivalent reflections. But where is the information to generate that asymmetry coming from? There is probably something I'm missing here, but i would definitely reprocess in the lower symmetry if the rotation angle was sufficient to give good completeness. eab - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFRfo5lUxlJ7aRr7hoRAkHvAJ9VRWDjbH8gcF1OW5vfprehPJDCUwCfTHoA nv3mtZH/Em2VlbDh9IFDvoU= =F/Om -END PGP SIGNATURE-
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
FYI, I do know of one example of a solved structure where some of the molecules in the ASU are poorly defined. In 1EKJ, 4 of the 8 molecules in the ASU have low B-factors (mid 30s) and 4 have high B-factors (50s-60s). In the unit cell these layers alternate. It is possible, if everything else has been excluded, that your crystal has a similar crystal-packing issue. 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 4/29/2013 9:30 AM, sonali dhindwal wrote: Dear Eleanor, Thanks for the suggestion, I just checked on the Zanuda program, it is also giving P1 as the best possible spacegroup for the molecule. and by not refining well, I meant for the electron density which is broken at many places at main chain, and poor electron density for the side chains which are otherwise good in other chains. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” *From:* Eleanor Dodson eleanor.dod...@york.ac.uk *To:* CCP4BB@JISCMAIL.AC.UK *Sent:* Monday, 29 April 2013 4:36 PM *Subject:* Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit There isnt much information here! Funny that 3 chains are poor - does that mean one and a half heterodimers? I presume you have checked spacegroup? Zanuda will test to see if any higher symmetry is present.. Eleanor On 29 April 2013 06:02, sonali dhindwal sonali11dhind...@yahoo.co.in mailto:sonali11dhind...@yahoo.co.in wrote: Dear All, We are working on a crystal structure with 12 molecules in an asymmetric unit. It is a heteromer and each chain is made up of two type of chains. Therefore there are 24 chains in an asymmetric unit. It is giving solution in P1. Rfactor and Rfree has reached 20.25 and 25.6 at a resolution of 1.8Angstrom All the chains are refining well and have good electron density except 3 chains which have very poor electron density in most of the region. Whereas, the crystal structure of variants of the same protein we have solved earlier refined well and has very good electron density in all the chains. What could be the possible reason for these three chains which are not refining properly. Any suggestions will be very useful. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.”
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
You may also get some insights from TLS refinement. Regards Thierry From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger Rowlett Sent: Monday, April 29, 2013 12:27 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit FYI, I do know of one example of a solved structure where some of the molecules in the ASU are poorly defined. In 1EKJ, 4 of the 8 molecules in the ASU have low B-factors (mid 30s) and 4 have high B-factors (50s-60s). In the unit cell these layers alternate. It is possible, if everything else has been excluded, that your crystal has a similar crystal-packing issue. 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.edumailto:rrowl...@colgate.edu On 4/29/2013 9:30 AM, sonali dhindwal wrote: Dear Eleanor, Thanks for the suggestion, I just checked on the Zanuda program, it is also giving P1 as the best possible spacegroup for the molecule. and by not refining well, I meant for the electron density which is broken at many places at main chain, and poor electron density for the side chains which are otherwise good in other chains. Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” From: Eleanor Dodson eleanor.dod...@york.ac.ukmailto:eleanor.dod...@york.ac.uk To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Sent: Monday, 29 April 2013 4:36 PM Subject: Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit There isnt much information here! Funny that 3 chains are poor - does that mean one and a half heterodimers? I presume you have checked spacegroup? Zanuda will test to see if any higher symmetry is present.. Eleanor On 29 April 2013 06:02, sonali dhindwal sonali11dhind...@yahoo.co.inmailto:sonali11dhind...@yahoo.co.in wrote: Dear All, We are working on a crystal structure with 12 molecules in an asymmetric unit. It is a heteromer and each chain is made up of two type of chains. Therefore there are 24 chains in an asymmetric unit. It is giving solution in P1. Rfactor and Rfree has reached 20.25 and 25.6 at a resolution of 1.8Angstrom All the chains are refining well and have good electron density except 3 chains which have very poor electron density in most of the region. Whereas, the crystal structure of variants of the same protein we have solved earlier refined well and has very good electron density in all the chains. What could be the possible reason for these three chains which are not refining properly. Any suggestions will be very useful. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” Notice: This e-mail message, together with any attachments, contains information of Merck Co., Inc. (One Merck Drive, Whitehouse Station, New Jersey, USA 08889), and/or its affiliates Direct contact information for affiliates is available at http://www.merck.com/contact/contacts.html) that may be confidential, proprietary copyrighted and/or legally privileged. It is intended solely for the use of the individual or entity named on this message. If you are not the intended recipient, and have received this message in error, please notify us immediately by reply e-mail and then delete it from your system.
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
Dear All, Thanks for the help and suggestion Francis, we used molrep for the structure solution. As I told that we already have the structure of the same protein, but a variant. So, we used its single chain (heteromer) as a model for molecular replacement. and Eleanor, I deleted once that chain and did refinement, all I was getting was the small small broken electron density everywhere in that chain region. Thanks again. -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” From: Francis E. Reyes francis.re...@colorado.edu To: sonali dhindwal sonali11dhind...@yahoo.co.in Sent: Monday, 29 April 2013 10:03 PM Subject: Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit Where are the phases coming from? MR? F On Apr 28, 2013, at 10:02 PM, sonali dhindwal sonali11dhind...@yahoo.co.in wrote: Dear All, We are working on a crystal structure with 12 molecules in an asymmetric unit. It is a heteromer and each chain is made up of two type of chains. Therefore there are 24 chains in an asymmetric unit. It is giving solution in P1. Rfactor and Rfree has reached 20.25 and 25.6 at a resolution of 1.8Angstrom All the chains are refining well and have good electron density except 3 chains which have very poor electron density in most of the region. Whereas, the crystal structure of variants of the same protein we have solved earlier refined well and has very good electron density in all the chains. What could be the possible reason for these three chains which are not refining properly. Any suggestions will be very useful. Thanks and Regards -- Sonali Dhindwal “Live as if you were to die tomorrow. Learn as if you were to live forever.” - Francis E. Reyes PhD 215 UCB University of Colorado at Boulder
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
On 04/29/13 12:26, Roger Rowlett wrote: FYI, I do know of one example of a solved structure where some of the molecules in the ASU are poorly defined. In 1EKJ, 4 of the 8 molecules in the ASU have low B-factors (mid 30s) and 4 have high B-factors (50s-60s). In the unit cell these layers alternate. It is possible, if everything else has been excluded, that your crystal has a similar crystal-packing issue. Another is 3HDH. Pig short chain L-3-hydroxyacyl-CoA dehydrongenase revisited: sequence analysis and crystal structure determination. Barycki, et al (1999) Protein Science 8: 2010-2018. Examination of the map in the region of subunit C revealed that the electron density was considerably weaker for the third subunit compared to the other subunits... -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Poor electron density in some of the chains in an asymmetric unit
and 2VAK...average Bs for the twelve, sequence identical, chains vary from 28 to 53. On 29 Apr 2013, at 22:09, David Schuller wrote: On 04/29/13 12:26, Roger Rowlett wrote: FYI, I do know of one example of a solved structure where some of the molecules in the ASU are poorly defined. In 1EKJ, 4 of the 8 molecules in the ASU have low B-factors (mid 30s) and 4 have high B-factors (50s-60s). In the unit cell these layers alternate. It is possible, if everything else has been excluded, that your crystal has a similar crystal-packing issue. Another is 3HDH. Pig short chain L-3-hydroxyacyl-CoA dehydrongenase revisited: sequence analysis and crystal structure determination. Barycki, et al (1999) Protein Science 8: 2010-2018. Examination of the map in the region of subunit C revealed that the electron density was considerably weaker for the third subunit compared to the other subunits... -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
[ccp4bb] Crystals in the news
http://news.discovery.com/earth/rocks-fossils/sea-squirt-crystal-solved-130429.htm Crystal Puzzle Solved with Sea Squirt RE vaterite Science 26 April 2013: 454-457.[DOI:10.1126/science.1232139] http://www.kare11.com/news/article/1023782/391/Strange-ice-phenomena-unfolds-at-Minn-lake Strange 'chandeliering ice' phenomena unfolds at Medicine Lake -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
