[ccp4bb] Postdoctoral position available at Caltech
The Hoelz laboratory at the California Institute of Technology seeks to recruit an outstanding postdoctoral scientist in structural biology. Our laboratory focuses on the structure-function relationships of multi-protein macromolecular assemblies. Major research themes are (1) the nuclear pore complex and evolutionarily-related vesicle coats, and (2) histone modifying enzymes and their resident multi-protein complexes. For recent publications, see, for example, Stavropoulos et al., NSMB, 2006; Hsia et al., CELL, 2007; Melcak et al., SCIENCE, 2007; Debler et al., MOL. CELL, 2008; Nagy et al., PNAS, 2009; Hsia et al., PNAS, 2010; Hoelz et el., Annu Rev. Biochem., 2011. This position requires a recent Ph.D. degree in biochemistry/biophysics with a strong background in x-ray crystallography. The successful applicant should be a highly motivated individual with good organizational, communication, and interpersonal skills, who is able to carry out a structure determination project from protein expression to structure refinement. The laboratory is newly constructed, well-funded, and well-equipped with modern biophysical instrumentation and current crystallization robotics. Importantly, Caltech has an exceptional infrastructure for macromolecular crystallography, including 40 % access to a high intensity, completely automated, state-of-the-art crystallography beamline equipped with a PILATUS detector at the Stanford Synchrotron Radiation Lightsource (SSRL). The position is available immediately and funded for 2+ years. Depending on performance, the position can be extended for up to five years. To apply, please send your CV, a statement of research interests, and the names and contact information of at least two references to André Hoelz (ho...@caltech.edu).
Re: [ccp4bb] SigmaA LABIN error
Hi Yogesh I was probably the last person to update the SIGMAA source code, but I have been using it without any problems. Could you post more info, specifically the shell script you are using and an mtzdump listing of the MTZ file header. If the problem can't be resolved from that info I may need to ask you for the MTZ file itself, if that's possible. Cheers -- Ian On Wed, May 18, 2011 at 10:45 PM, Yogesh Gupta wrote: > My goal is to combine experimental phases to calculated phases from partial > model. I am using SIGMAA in CCP4, which gave the following error, all labels > that program can not find are present in the input file though. > > Error in LABIN: label PHIB not found in file! > Error in LABIN: label FOM not found in file! > Error in LABIN: label HLA not found in file! > Error in LABIN: label HLB not found in file! > Error in LABIN: label HLC not found in file! > Error in LABIN: label HLD not found in file! > Error in LABIN: label FB not found in file! > SIGMAA: Error in label assignments in LABIN > > Could someone help me fix this or point me to other program that could do > the phase combination similar to SIGMAA? > > Thanks for your time. > > -Yogesh > > -- > -- > Yogesh K. Gupta, Ph.D. > Dept of Structural & Chemical Biology > Mount Sinai School of Medicine > Icahn Medical Institute > 1 Gustave L. Levy Place, Box 1677 > New York, NY, USA 10029 > > Tel:+1 212-659-8639 > -- >
[ccp4bb] Twinning, Wilson scaling and B factor
Dear ccp4 users, I have a data set arising from a nearly-perfect pseudo-merohedrally twinned cystal, diffracting up to 3 A. I solved the structure and ready for deposition, but there is still a trouble. The Wilson scaling from raw data gave a B of 3A^2. Initially, I did not seemed too alarming. But I do not know why I have these statistics. Does anyone know why Wilson scaling falls when treating that kind of twinned data? I read that twinned data do not obey twe Wilson statistics but I don't know why. Here the presentation I read: http://bstr521.biostr.washington.edu/PDF/Twinning_2007.pdf Do you know any articles, reviews or book in which this particular aspect of of twinned data is treated in depth, possibly in mathematical manner? Thanks to all Fulvio Saccoccia, PhD student Biochemical Sciences Dept. Sapienza University of Rome
[ccp4bb] Low resolution refinement
Dear all I am refining a structure at 3.4 A resolution that contains 3 molecules in the a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric functional structure expected for this class of proteins. The other two chains B and C, which also form the functional dimer, seem to be, somehow, a lot more flexible than chain A. As a result, whereas the electron density map, b-factor and geometry for chain A is pretty reasonable for a 3.4 A resolution structure, the refinement for the other two chains (B and C) does not behave well. Even playing with different weights for geometry, analysing different levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map for the helical regions is ok, but the electron density map for strands and loops of chains B and C are broken along the main chain, B-factors are really high, and the geometry keeps being distorted. Right now, the R-factor and R-free are 24.2 and 28.6, respectively. Any suggestions in how to proceed the refinement? And even a more difficult question, how do we report this type of structure? How do we deposit those coordinates? We can certainly use chain A as a model to perform interesting studies of structure-function relationship, but we know that chain B and chain C have problems. Any help will be greatly appreciated. Regards Joane -- Joane Kathelen Rustiguel Bonalumi Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP Laboratório de Cristalografia de Proteínas Departamento de Física e Química Fone: +55.16.3602.4193
Re: [ccp4bb] Twinning, Wilson scaling and B factor
Hi Fulvio There are 2 different issues here: the Wilson plot scale & B factor on the one hand and Wilson statistics on the other. The first are not affected by twinning since they depend only on the intensity averages in shells. The second refers to the distribution of intensities (i.e. the proportion of reflections with intensity less than a specified value) within a shell, or to the distribution of normalised intensities (Z = I/ ignoring symmetry issues for now) over the whole dataset. This distribution is different for a twin because averaging the components which contribute to the intensity of a twinned reflection tends to shift the distribution towards the mean, so you get fewer extreme values. The Wilson B factor is not a 'statistic' in the strict sense, merely a derived parameter. I suspect the low value you get has more to do with the fact that the resolution is only 3 A, than the fact it's twinned. See here for more mathematically-oriented info: http://www.ccp4.ac.uk/dist/html/pxmaths/bmg10.html Cheers -- Ian On Thu, May 19, 2011 at 1:45 PM, fulvio saccoccia wrote: > Dear ccp4 users, > I have a data set arising from a nearly-perfect pseudo-merohedrally > twinned cystal, diffracting up to 3 A. I solved the structure and ready > for deposition, but there is still a trouble. > The Wilson scaling from raw data gave a B of 3A^2. > Initially, I did not seemed too alarming. But I do not know why I have > these statistics. > > Does anyone know why Wilson scaling falls when treating that kind of > twinned data? I read that twinned data do not obey twe Wilson statistics > but I don't know why. > Here the presentation I read: > > http://bstr521.biostr.washington.edu/PDF/Twinning_2007.pdf > > Do you know any articles, reviews or book in which this particular > aspect of of twinned data is treated in depth, possibly in mathematical > manner? > > Thanks to all > > Fulvio Saccoccia, PhD student > Biochemical Sciences Dept. > Sapienza University of Rome >
Re: [ccp4bb] Twinning, Wilson scaling and B factor
Ok, so another question in my mind: the data come from a collection interrupted after the first 200 frames and restarted after 1 hour to recover the missed 160 frames. The first and second group of frames, if separately indexed, give cell parameter values that differ of about 1A for axes and 1 degree in angle. Can this affect thw wilson plot scaline and as consequence the B factor? Il giorno gio, 19/05/2011 alle 14.14 +0100, Ian Tickle ha scritto: > Hi Fulvio > > There are 2 different issues here: the Wilson plot scale & B factor on > the one hand and Wilson statistics on the other. The first are not > affected by twinning since they depend only on the intensity averages > in shells. The second refers to the distribution of intensities (i.e. > the proportion of reflections with intensity less than a specified > value) within a shell, or to the distribution of normalised > intensities (Z = I/ ignoring symmetry issues for now) over the > whole dataset. This distribution is different for a twin because > averaging the components which contribute to the intensity of a > twinned reflection tends to shift the distribution towards the mean, > so you get fewer extreme values. > > The Wilson B factor is not a 'statistic' in the strict sense, merely a > derived parameter. I suspect the low value you get has more to do > with the fact that the resolution is only 3 A, than the fact it's > twinned. > > See here for more mathematically-oriented info: > > http://www.ccp4.ac.uk/dist/html/pxmaths/bmg10.html > > Cheers > > -- Ian > > On Thu, May 19, 2011 at 1:45 PM, fulvio saccoccia > wrote: > > Dear ccp4 users, > >I have a data set arising from a nearly-perfect pseudo-merohedrally > > twinned cystal, diffracting up to 3 A. I solved the structure and ready > > for deposition, but there is still a trouble. > > The Wilson scaling from raw data gave a B of 3A^2. > > Initially, I did not seemed too alarming. But I do not know why I have > > these statistics. > > > > Does anyone know why Wilson scaling falls when treating that kind of > > twinned data? I read that twinned data do not obey twe Wilson statistics > > but I don't know why. > > Here the presentation I read: > > > > http://bstr521.biostr.washington.edu/PDF/Twinning_2007.pdf > > > > Do you know any articles, reviews or book in which this particular > > aspect of of twinned data is treated in depth, possibly in mathematical > > manner? > > > > Thanks to all > > > > Fulvio Saccoccia, PhD student > > Biochemical Sciences Dept. > > Sapienza University of Rome > >
Re: [ccp4bb] Twinning, Wilson scaling and B factor
Hi Fulvio, Yes, it can. An increase in cell parameters can indicate a failure of the cooling system or (more likely) radiation damage of the crystal. For refinement you probably do not need all those 360 frames, unless you collected thin frames and your space group is P1 I suggest you integrate the two sets separately and check how well they merge - does the Rmeas rise a lot? Then it may be worth using only the first data set (unless your data are incomplete). Tim On Thu, May 19, 2011 at 04:16:22PM +0200, fulvio saccoccia wrote: > Ok, so another question in my mind: > the data come from a collection interrupted after the first 200 frames > and restarted after 1 hour to recover the missed 160 frames. > The first and second group of frames, if separately indexed, give cell > parameter values that differ of about 1A for axes and 1 degree in angle. > Can this affect thw wilson plot scaline and as consequence the B > factor? > > > Il giorno gio, 19/05/2011 alle 14.14 +0100, Ian Tickle ha scritto: > > Hi Fulvio > > > > There are 2 different issues here: the Wilson plot scale & B factor on > > the one hand and Wilson statistics on the other. The first are not > > affected by twinning since they depend only on the intensity averages > > in shells. The second refers to the distribution of intensities (i.e. > > the proportion of reflections with intensity less than a specified > > value) within a shell, or to the distribution of normalised > > intensities (Z = I/ ignoring symmetry issues for now) over the > > whole dataset. This distribution is different for a twin because > > averaging the components which contribute to the intensity of a > > twinned reflection tends to shift the distribution towards the mean, > > so you get fewer extreme values. > > > > The Wilson B factor is not a 'statistic' in the strict sense, merely a > > derived parameter. I suspect the low value you get has more to do > > with the fact that the resolution is only 3 A, than the fact it's > > twinned. > > > > See here for more mathematically-oriented info: > > > > http://www.ccp4.ac.uk/dist/html/pxmaths/bmg10.html > > > > Cheers > > > > -- Ian > > > > On Thu, May 19, 2011 at 1:45 PM, fulvio saccoccia > > wrote: > > > Dear ccp4 users, > > >I have a data set arising from a nearly-perfect pseudo-merohedrally > > > twinned cystal, diffracting up to 3 A. I solved the structure and ready > > > for deposition, but there is still a trouble. > > > The Wilson scaling from raw data gave a B of 3A^2. > > > Initially, I did not seemed too alarming. But I do not know why I have > > > these statistics. > > > > > > Does anyone know why Wilson scaling falls when treating that kind of > > > twinned data? I read that twinned data do not obey twe Wilson statistics > > > but I don't know why. > > > Here the presentation I read: > > > > > > http://bstr521.biostr.washington.edu/PDF/Twinning_2007.pdf > > > > > > Do you know any articles, reviews or book in which this particular > > > aspect of of twinned data is treated in depth, possibly in mathematical > > > manner? > > > > > > Thanks to all > > > > > > Fulvio Saccoccia, PhD student > > > Biochemical Sciences Dept. > > > Sapienza University of Rome > > > -- -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen phone: +49 (0)551 39 22149 GPG Key ID = A46BEE1A signature.asc Description: Digital signature
Re: [ccp4bb] Low resolution refinement
Hi Joane, I'm not sure if I understand your description of the structure (chain A forms the dimer? Does it form a dimer with a crystallographically related chain A?) . But anyway, I don't think you need to worry. Your R/Rfree are excellent for this resolution. There's not much you can do about the fact that some molecules in the AU are less ordered; this happens fairly often. As you say, you can use the best ordered chain as a representative model. Refine everything as well as you can and move on I would say. Bert On 5/19/11 9:02 AM, "Joane Kathelen Rustiguel" wrote: Dear all I am refining a structure at 3.4 A resolution that contains 3 molecules in the a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric functional structure expected for this class of proteins. The other two chains B and C, which also form the functional dimer, seem to be, somehow, a lot more flexible than chain A. As a result, whereas the electron density map, b-factor and geometry for chain A is pretty reasonable for a 3.4 A resolution structure, the refinement for the other two chains (B and C) does not behave well. Even playing with different weights for geometry, analysing different levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map for the helical regions is ok, but the electron density map for strands and loops of chains B and C are broken along the main chain, B-factors are really high, and the geometry keeps being distorted. Right now, the R-factor and R-free are 24.2 and 28.6, respectively. Any suggestions in how to proceed the refinement? And even a more difficult question, how do we report this type of structure? How do we deposit those coordinates? We can certainly use chain A as a model to perform interesting studies of structure-function relationship, but we know that chain B and chain C have problems. Any help will be greatly appreciated. Regards Joane -- Joane Kathelen Rustiguel Bonalumi Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP Laboratório de Cristalografia de Proteínas Departamento de Física e Química Fone: +55.16.3602.4193
Re: [ccp4bb] Low resolution refinement
One more thing though: have you refined with the NCS restraints off or on? Presumably on, seeing that you have a small gap between R and Rfree? It may be worth turning the restraints off or modify the NCS selections: if your 3 molecules are substantially different, applying NCS may make things worse rather than better (ie you're averaging things that are different). Good luck, Bert On 5/19/11 9:02 AM, "Joane Kathelen Rustiguel" wrote: Dear all I am refining a structure at 3.4 A resolution that contains 3 molecules in the a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric functional structure expected for this class of proteins. The other two chains B and C, which also form the functional dimer, seem to be, somehow, a lot more flexible than chain A. As a result, whereas the electron density map, b-factor and geometry for chain A is pretty reasonable for a 3.4 A resolution structure, the refinement for the other two chains (B and C) does not behave well. Even playing with different weights for geometry, analysing different levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map for the helical regions is ok, but the electron density map for strands and loops of chains B and C are broken along the main chain, B-factors are really high, and the geometry keeps being distorted. Right now, the R-factor and R-free are 24.2 and 28.6, respectively. Any suggestions in how to proceed the refinement? And even a more difficult question, how do we report this type of structure? How do we deposit those coordinates? We can certainly use chain A as a model to perform interesting studies of structure-function relationship, but we know that chain B and chain C have problems. Any help will be greatly appreciated. Regards Joane -- Joane Kathelen Rustiguel Bonalumi Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP Laboratório de Cristalografia de Proteínas Departamento de Física e Química Fone: +55.16.3602.4193
Re: [ccp4bb] Low resolution refinement
Dear Joane, Bert's recommendations are very good, but I'd like to add a little caution. If just part of the molecule has bad density then it is not unusual, but if the whole chain does not look very good (missing side-chains or backbone) you may have a couple of things going on. First, I would first suspect NCS issues (as Bert has) where you might be trying to force too high a space group since an NCS operator might be darn close to a crystallographic operator. Second it could also be caused by twinning, but since your R-factors are pretty respectable I would imagine that is probably not the case although you may want to look into it. Third, it could be static disorder where the crappy density might be two molecules on top of each other but one is slightly shifted. A way to tell is to scale down in your electron density maps to see if there are extra strands and helices next to the strands and helices you have built. Hopefully this is not the case and it is just NCS messing with you... Jon -- Jonathan P. Schuermann, Ph. D. Beamline Scientist NE-CAT, Building 436E Advanced Photon Source (APS) Argonne National Laboratory 9700 South Cass Avenue Argonne, IL 60439 email: schue...@anl.gov Tel: (630) 252-0682 Fax: (630) 252-0687 On 05/19/2011 10:06 AM, Van Den Berg, Bert wrote: One more thing though: have you refined with the NCS restraints off or on? Presumably on, seeing that you have a small gap between R and Rfree? It may be worth turning the restraints off or modify the NCS selections: if your 3 molecules are substantially different, applying NCS may make things worse rather than better (ie you're averaging things that are different). Good luck, Bert On 5/19/11 9:02 AM, "Joane Kathelen Rustiguel" wrote: Dear all I am refining a structure at 3.4 A resolution that contains 3 molecules in the a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric functional structure expected for this class of proteins. The other two chains B and C, which also form the functional dimer, seem to be, somehow, a lot more flexible than chain A. As a result, whereas the electron density map, b-factor and geometry for chain A is pretty reasonable for a 3.4 A resolution structure, the refinement for the other two chains (B and C) does not behave well. Even playing with different weights for geometry, analysing different levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map for the helical regions is ok, but the electron density map for strands and loops of chains B and C are broken along the main chain, B-factors are really high, and the geometry keeps being distorted. Right now, the R-factor and R-free are 24.2 and 28.6, respectively. Any suggestions in how to proceed the refinement? And even a more difficult question, how do we report this type of structure? How do we deposit those coordinates? We can certainly use chain A as a model to perform interesting studies of structure-function relationship, but we know that chain B and chain C have problems. Any help will be greatly appreciated. Regards Joane -- Joane Kathelen Rustiguel Bonalumi Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP Laboratório de Cristalografia de Proteínas Departamento de Física e Química Fone: +55.16.3602.4193
Re: [ccp4bb] Low resolution refinement
Missing density could point to incomplete data, how is the completeness at high and low resolution? Sent from my iPhone On May 19, 2011, at 11:54 AM, Jon Schuermann wrote: > Dear Joane, > >Bert's recommendations are very good, but I'd like to add a little > caution. If just part of the molecule has bad density then it is not unusual, > but if the whole chain does not look very good (missing side-chains or > backbone) you may have a couple of things going on. First, I would first > suspect NCS issues (as Bert has) where you might be trying to force too high > a space group since an NCS operator might be darn close to a crystallographic > operator. Second it could also be caused by twinning, but since your > R-factors are pretty respectable I would imagine that is probably not the > case although you may want to look into it. Third, it could be static > disorder where the crappy density might be two molecules on top of each other > but one is slightly shifted. A way to tell is to scale down in your electron > density maps to see if there are extra strands and helices next to the > strands and helices you have built. Hopefully this is not the case and it is > just NCS messing with you... > > Jon > > -- > Jonathan P. Schuermann, Ph. D. > Beamline Scientist > NE-CAT, Building 436E > Advanced Photon Source (APS) > Argonne National Laboratory > 9700 South Cass Avenue > Argonne, IL 60439 > > email: schue...@anl.gov > Tel: (630) 252-0682 > Fax: (630) 252-0687 > > > On 05/19/2011 10:06 AM, Van Den Berg, Bert wrote: >> >> One more thing though: have you refined with the NCS restraints off or on? >> Presumably on, seeing that you have a small gap between R and Rfree? It may >> be worth turning the restraints off or modify the NCS selections: if your 3 >> molecules are substantially different, applying NCS may make things worse >> rather than better (ie you’re averaging things that are different). >> >> Good luck, Bert >> >> >> On 5/19/11 9:02 AM, "Joane Kathelen Rustiguel" wrote: >> >> Dear all >> >> >> I am refining a structure at 3.4 A resolution that contains 3 molecules in >> the >> a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric >> functional structure expected for this class of proteins. The other two >> chains >> B and C, which also form the functional dimer, seem to be, somehow, a lot >> more >> flexible than chain A. As a result, whereas the electron density map, >> b-factor >> and geometry for chain A is pretty reasonable for a 3.4 A resolution >> structure, the refinement for the other two chains (B and C) does not behave >> well. Even playing with different weights for geometry, analysing different >> levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map >> for the helical regions is ok, but the electron density map for strands and >> loops of chains B and C are broken along the main chain, B-factors are really >> high, and the geometry keeps being distorted. >> >> Right now, the R-factor and R-free are 24.2 and 28.6, respectively. >> >> Any suggestions in how to proceed the refinement? >> And even a more difficult question, how do we report this type of structure? >> How do we deposit those coordinates? We can certainly use chain A as a model >> to perform interesting studies of structure-function relationship, but we >> know >> that chain B and chain C have problems. >> >> Any help will be greatly appreciated. >> >> Regards >> >> Joane >> >> >> -- >> Joane Kathelen Rustiguel Bonalumi >> Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP >> Laboratório de Cristalografia de Proteínas >> Departamento de Física e Química >> Fone: +55.16.3602.4193 >> >> > >
[ccp4bb] PhD or postdoctoral fellowships at the Max Planck Institute in Tuebingen, Germany
Max Planck Institute for Developmental Biology Tuebingen, Germany. PhD or postdoctoral positions funded by tax free fellowships are available in the research group of Remco Sprangers and in the research group of Silke Wiesner. We are looking for candidates that have a strong interest structural biology and biochemistry. More details about the projects that combine NMR spectroscopy, X-ray crystallography and biochemical approaches can be found below. Both laboratories are very well equipped, including new 600 and 800 MHz NMR spectrometers, crystallization robots and state-of-the-art instrumentation for protein expression and purification. In addition, we have regular access to the synchrotron facility at the SLS. Applicants for the PhD positions should have a strong background in biochemistry, chemistry and/or biophysics. Applicants for the postdoctoral positions should have a PhD in NMR spectroscopy or X-ray crystallography. Please contact remco.sprang...@tuebingen.mpg.de or silke.wies...@tuebingen.mpg.de by June 15, 2011 and include a pdf file with a brief CV, names of two referees and a summary of research interests and previous research projects. The research group of Remco Sprangers (http://www.eb.tuebingen.mpg.de/research-groups/remco-sprangers) studies how protein motions determine enzymatic function. We are especially interested in the machinery that degrades mRNA. Structural changes in the degradation complex allow for a tight regulation of the mRNA decay process that is crucial for cellular homeostasis. To understand how this is achieved on an atomic level, we use novel techniques in high resolution NMR spectroscopy (methyl TROSY) in combination with X-ray crystallography and biochemical experiments. The research group of Silke Wiesner (http://www.eb.tuebingen.mpg.de/research-groups/silke-wiesner) is interested in the structure, function, and regulation of the ubiquitination machinery. Ubiquitination marks proteins for degradation and thereby controls cellular signaling and behavior. We seek to provide a molecular basis for protein ubiquitination and to understand how diseases like cancer arise from dysfunctional ubiquitination enzymes. We gain structural information from NMR and/or X-ray crystallography and combine this information with investigations targeted at functional aspects using both in vitro and in vivo studies.
Re: [ccp4bb] Low resolution refinement
This reminds me of: Pig heart short chain L-3-hydroxyacyl-CoA dehydrogenase revisited: Sequence analysis and crystal structure determination Barycki JJ, O'Brien LK, Birktoft JJ, Strauss AW, Banaszak LJ Protein Science (Oct 1999) Vol 8, pp 2010-2018. In which the protein in question also had one monomer forming a dimer about a crystallographic axis, and two monomers forming a dimer elsewhere in the asymmetric unit. A portion of the molecule had messy density, which caused difficulties literally for decades. The eventual solution was to switch to a different species (human). After which, a MR solution of the original porcine data was possible. I believe the disorder was judged to be intrinsic. On 05/19/11 09:02, Joane Kathelen Rustiguel wrote: Dear all I am refining a structure at 3.4 A resolution that contains 3 molecules in the a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric functional structure expected for this class of proteins. The other two chains B and C, which also form the functional dimer, seem to be, somehow, a lot more flexible than chain A. As a result, whereas the electron density map, b-factor and geometry for chain A is pretty reasonable for a 3.4 A resolution structure, the refinement for the other two chains (B and C) does not behave well. Even playing with different weights for geometry, analysing different levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map for the helical regions is ok, but the electron density map for strands and loops of chains B and C are broken along the main chain, B-factors are really high, and the geometry keeps being distorted. Right now, the R-factor and R-free are 24.2 and 28.6, respectively. Any suggestions in how to proceed the refinement? And even a more difficult question, how do we report this type of structure? How do we deposit those coordinates? We can certainly use chain A as a model to perform interesting studies of structure-function relationship, but we know that chain B and chain C have problems. Any help will be greatly appreciated. Regards Joane -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] Low resolution refinement
Dear Joane, We have had very good luck refining low resolution structures with dramatic improvement using several newish options in refmac - particularly when NCS is present! Those options are jelly body restraints, automatic NCS restraints, and map sharpening. Here is a description cut-and-pasted from Garib's presentation (www.ccp4.ac.uk/schools/China-2011/tutorials/refmac_tutorial.pdf) V) Low resolution refinement If you have older version of ccp4 then you can follow the instructions described in the presentation: www.ysbl.york.ac.uk/refmac/Presentations/Refmac_Erice_workshop.ppt slides 45-50 Full description of new features are in: www.ysbl.york.ac.uk/refmac/data/refmac_news.html In the new version of ccp4 there are options for jelly body, automatic NCS and map sharpening. a) Jelly body is under “Refinement Parameters”. You need to click “Use jelly- body refinement with sigma”. Change sigma to 0.01 or 0.02. This value defines “jelliness”. Smaller value means tighter restraints b) Automatic NCS restraints are under “Setup Non-Crystallographic Symmetry”. You need to cick “use automatically generated local NCS restraints”. You can also use global NCS c) Map sharpening is under “Monitoring and Output Options”. You need to click “Perform map sharpening with B value 20.0”. B value should a little bit smaller than Wilson’s B value. more in depth information can be found here: www.ccp4.ac.uk/schools/China-2011/talks/refmac_Shanghai.pdf This is definitely something you should explore. good luck, Eric Eric T. Larson, PhD Biomolecular Structure Center Department of Biochemistry Box 357742 University of Washington Seattle, WA 98195 email: larso...@u.washington.edu On Thu, 19 May 2011, David Schuller wrote: > This reminds me of: > > Pig heart short chain L-3-hydroxyacyl-CoA dehydrogenase revisited: Sequence > analysis and crystal structure determination > Barycki JJ, O'Brien LK, Birktoft JJ, Strauss AW, Banaszak LJ > Protein Science (Oct 1999) Vol 8, pp 2010-2018. > > In which the protein in question also had one monomer forming a dimer about a > crystallographic axis, and two monomers forming a dimer elsewhere in the > asymmetric unit. > > A portion of the molecule had messy density, which caused difficulties > literally for decades. The eventual solution was to switch to a different > species (human). After which, a MR solution of the original porcine data was > possible. I believe the disorder was judged to be intrinsic. > > > > On 05/19/11 09:02, Joane Kathelen Rustiguel wrote: >> Dear all >> >> >> I am refining a structure at 3.4 A resolution that contains 3 molecules in >> the >> a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric >> functional structure expected for this class of proteins. The other two >> chains >> B and C, which also form the functional dimer, seem to be, somehow, a lot >> more >> flexible than chain A. As a result, whereas the electron density map, >> b-factor >> and geometry for chain A is pretty reasonable for a 3.4 A resolution >> structure, the refinement for the other two chains (B and C) does not behave >> well. Even playing with different weights for geometry, analysing different >> levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map >> for the helical regions is ok, but the electron density map for strands and >> loops of chains B and C are broken along the main chain, B-factors are >> really >> high, and the geometry keeps being distorted. >> >> Right now, the R-factor and R-free are 24.2 and 28.6, respectively. >> >> Any suggestions in how to proceed the refinement? >> And even a more difficult question, how do we report this type of structure? >> How do we deposit those coordinates? We can certainly use chain A as a model >> to perform interesting studies of structure-function relationship, but we >> know >> that chain B and chain C have problems. >> >> Any help will be greatly appreciated. >> >> Regards >> >> Joane >> >> > > > -- > === > All Things Serve the Beam > === > David J. Schuller > modern man in a post-modern world > MacCHESS, Cornell University > schul...@cornell.edu >
[ccp4bb] One Postdoc Position Available in Structural Virology
Applications are invited for a postdoctoral position in Dr. Fang Li's lab at the Department of Pharmacology, University of Minnesota Twin Cities. Research involves biochemical and structural studies on proteins that guide invasion and replication of important viral pathogens. For more information about Dr. Li's lab, visit http://www.msi.umn.edu/~lifang/. Candidates should be highly motivated about scientific research and must have completed PhD at the time of the appointment. Strong background in X-ray crystallography and protein biochemistry is required. Interested applicants should send (1) a resume, (2) a one-page summary of previous research experience, and (3) arrange two letters of recommendation to be sent to Dr. Fang Li via emai: lif...@umn.edu
