Re: [ccp4bb] Set up ccp4 environment
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Uma, you have to run the command source /xtal/Suites/CCP4/ccp4-6.4.0/bin/ccp4.setup-sh in the terminal from which you start xdsstat (and f2mtz etc.pp). It is best to place the above command into your file ~/.bashrc so that you do not need to type it each time you want to use ccp4. (you will have to adjust the path /xtal/Suites/CCP4/ccp4-6.4.0 according to your installation). The file .bashrc resides in your home directory. Best, Tim On 03/11/2014 10:37 PM, Uma Ratu wrote: Dear All: I try to run xds in linux, but have some problems. With xdsconv, it complains: f2mtz: error while loading shared libraries: libccp4f.so.0: cannot open shared object file: No such file or directory cad: error while loading shared libraries: libccp4f.so.0: cannot open shared object file: No such file or directory With xdsstat, it complains: xdsstat: Cannot open environ.def It seems that one needs to set up a CCP4 environment in order to run xds in linux. I have ccp4 (the latest vision for linux) installed. And I use Ubuntu 12.04 LTS. Thank you for your advice Uma - -- - -- 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 Icedove - http://www.enigmail.net/ iD8DBQFTICwKUxlJ7aRr7hoRAj/tAJ0TvaJsyyqpAi3l/bg3oT4suzm/EACfV9KK 5IEXBIUDbUM2fbbEF3QY4PQ= =rTR6 -END PGP SIGNATURE-
Re: [ccp4bb] Set up ccp4 environment
Dear Tim: With the commend of source /xtal/Suites/CCP4/ccp4-6.4.0/bin/ccp4.setup-sh, I am able to run the xdsstat and f2mtz now. Thank you very much for your help! Uma On Wed, Mar 12, 2014 at 5:42 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Uma, you have to run the command source /xtal/Suites/CCP4/ccp4-6.4.0/bin/ccp4.setup-sh in the terminal from which you start xdsstat (and f2mtz etc.pp). It is best to place the above command into your file ~/.bashrc so that you do not need to type it each time you want to use ccp4. (you will have to adjust the path /xtal/Suites/CCP4/ccp4-6.4.0 according to your installation). The file .bashrc resides in your home directory. Best, Tim On 03/11/2014 10:37 PM, Uma Ratu wrote: Dear All: I try to run xds in linux, but have some problems. With xdsconv, it complains: f2mtz: error while loading shared libraries: libccp4f.so.0: cannot open shared object file: No such file or directory cad: error while loading shared libraries: libccp4f.so.0: cannot open shared object file: No such file or directory With xdsstat, it complains: xdsstat: Cannot open environ.def It seems that one needs to set up a CCP4 environment in order to run xds in linux. I have ccp4 (the latest vision for linux) installed. And I use Ubuntu 12.04 LTS. Thank you for your advice Uma - -- - -- 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 Icedove - http://www.enigmail.net/ iD8DBQFTICwKUxlJ7aRr7hoRAj/tAJ0TvaJsyyqpAi3l/bg3oT4suzm/EACfV9KK 5IEXBIUDbUM2fbbEF3QY4PQ= =rTR6 -END PGP SIGNATURE-
[ccp4bb] crystallography position open at Evotec
Dear All, We have a great opportunity for somebody to join the structural biology team at Evotec as a crystallographer. The closing date for applications is the 31st March. Please make applications via the website as listed in the advert below. Best regards, Paul.. Crystallographer Would you like to work in exciting, cutting edge research? Why not join the X-Ray Crystallographers within the Structural Biology Department at Evotec (UK) Ltd Location: Oxfordshire, UK Full time; Permanent Salary: Competitive Evotec (UK) Ltd is currently seeking X-Ray Crystallographers for our Structural Biology Department based in Oxfordshire. X-Ray Crystallography works closely with our Discovery Chemistry Department and with clients to develop novel small molecule drugs. The group is at the forefront of new science and technology, and is seeking to support its continued expansion. The successful candidates will be part of the Structural Biology group responsible for expression, purification, crystallisation and structure determination of proteins and protein-ligand complexes. Desirable Knowledge, Skills and Abilities: * Knowledge of state of the art crystallographic methods * Skills in molecular biology and protein purification * Expertise in protein-ligand complex crystallisation problems would be an advantage though not essential * Excellent communication and interpersonal skills * Client interaction and project management skills Education and Experience: * PhD qualified or equivalent in Chemistry, Biochemistry, Molecular Biology or Biophysics * Experience in protein X-ray crystallography * Candidates will also be considered who have a Masters degree with extensive experience or equivalent We offer competitive salaries plus extensive benefits including annual bonus, pension plan, private medical and dental cover. If you feel that your skills and experience match what we are looking for, please apply via the website: http://www.evotec.com http://www.evotec.com Dr. Paul A. McEwan Senior Scientist I, Structural Biology +44. (0)1235 838802 +44. (0)1235 863139 (Fax) paul.mce...@evotec.com mailto:paul.mce...@evotec.com www.evotec.com http://www.evotec.com/ Evotec (UK) Ltd. 114 Innovation Drive Milton Park Abingdon Oxfordshire OX14 4RZ (United Kingdom) Evotec (UK) Ltd is a limited company registered in England and Wales. Registration number:2674265. Registered office: 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire, OX14 4RZ, United Kingdom. image001.pngimage002.png
[ccp4bb] account creation for CCP4wiki (and XDSwiki)
Dear all, today we re-opened account creation for CCP4wiki (and XDSwiki). It had been closed since we were hit by spammers (like e.g. PyMolwiki), but we recently discovered a method that helps to prove that the account is really created by a a human being - as opposed to a computer program that creates spam articles with e.g. Viagra advertisements. Account creation now uses a Captcha which is not of the boring type, but rather challenges your expertise in telling cats from dogs. Happy wiki-editing! Kay smime.p7s Description: S/MIME Cryptographic Signature
Re: [ccp4bb] twinning problem ?
Zbyszek - do you have any measure of unintegrated streaks? It could be a help to at least have a rough score. Eleanor On 11 March 2014 20:04, Zbyszek Otwinowski zbys...@work.swmed.edu wrote: Shape of the diffraction spots changes in the statistical disorder -- twinning continuum. At both ends spots shape is like in diffraction from crystals without such disorder. However, in the intermediate case, electron density autocorrelation function has additional component to one resulting from ordered crystal. This additional component of autocorrelation creates characteristic non-Bragg diffraction, e.g. streaks aligned with particular unit cell axis. In the absence of such diffraction pattern, the ambiguity is binary. The description of the problem indicates statistical disorder. Zbyszek Otwinowski Hi, If there's an NCS translation, recent versions of Phaser can account for it and give moment tests that can detect twinning even in the presence of tNCS. But I agree with Eleanor that the L test is generally a good choice in these cases. However, the fact that you see density suggests that your crystal might be more on the statistical disorder side of the statistical disorder -- twinning continuum, i.e. the crystal doesn't have mosaic blocks corresponding to one twin fraction that are large compared to the coherence length of the X-rays. So you might want to try refinement with the whole structure duplicated as alternate conformers. Best wishes, Randy Read - Randy J. Read Department of Haematology, University of Cambridge Cambridge Institute for Medical ResearchTel: +44 1223 336500 Wellcome Trust/MRC Building Fax: +44 1223 336827 Hills Road E-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 11 Mar 2014, at 14:10, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: Sorry - hadnt finished.. The twinning tests are distorted by NC translation - usually the L test is safe, but the others are all suspect.. On 11 March 2014 14:09, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: What is the NC translation? If there is a factor of 0.5 that makes SG determination complicated.. Eleanor On 11 March 2014 14:04, Stephen Cusack cus...@embl.fr wrote: Dear All, I have 2.6 A data and unambiguous molecular replacement solution for two copies/asymmetric unit of a 80 K protein for a crystal integrated in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. Refinement allowed rebuilding/completion of the model in the noraml way but the R-free does not go below 30%. The map in the model regions looks generally fine but there is a lot of extra positive density in the solvent regions (some of it looking like weak density for helices and strands) and unexpected positive peaks within the model region. Careful inspection allowed manual positioning of a completely different, overlapping solution for the dimer which fits the extra density perfectly. The two incompatible solutions are related by a 2-fold axis parallel to a. This clearly suggests some kind of twinning. However twinning analysis programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of pseudo-merohedral twinning (-h, l, k) do not reveal any significant twinning fraction and proclaim the data likely to be untwinned. (NB. The programmes do however highlight a non-crystallographic translation and there are systematic intensity differences in the data). Refinement, including this twinning law made no difference since the estimated twinning fraction was 0.02. Yet the extra density is clearly there and I know exactly the real-space transformation between the two packing solutions. How can I best take into account this alternative solution (occupancy seems to be around 20-30%) in the refinement ? thanks for your suggestions Stephen -- ** Dr. Stephen Cusack, Head of Grenoble Outstation of EMBL Group leader in structural biology of protein-RNA complexes and viral proteins Joint appointment in EMBL Genome Biology Programme Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell Interactions (UVHCI) ** Email: cus...@embl.fr Website: http://www.embl.fr Tel:(33) 4 76 20 7238Secretary (33) 4 76 20 7123 Fax:(33) 4 76 20 7199 Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, 6 Rue Jules Horowitz, 38042 Grenoble, France ** Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX
Re: [ccp4bb] account creation for CCP4wiki (and XDSwiki)
On 03/12/14 11:52, Kay Diederichs wrote: ... Account creation now uses a Captcha which is not of the boring type, but rather challenges your expertise in telling cats from dogs. I have heard that this algorithm can be fooled by cosmetic surgery. http://www.condenaststore.com/-sp/No-Caption-Advertisement-for-cosmetic-surgeon-There-s-a-picture-of-a-dog-New-Yorker-Cartoon-Prints_i8542378_.htm -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
Re: [ccp4bb] twinning problem ?
Dear Stephen, I have seen a similar effect in the structure of F1-ATPase complexed with the full length inhibitor protein. The inhibitor is a dimer, and it actually couples 2 copies of the ATPase, but it crystallised with only one copy of the ATPase per asymmetric unit. When I solved the structure by MR, I saw additional density that could not be accounted for. The extra density was, in fact, a second ATPase molecule that was related to the first by a 120 degree rotation about the pseudo 3-fold axis of the enzyme. The dimers were packing with statistical disorder in the crystal lattice. This gave rise to clear streaking between Bragg spots in the diffraction images in a direction that was consistent with that expected from the statistical packing of the inhibitor linked dimers. Two copies of F1 were included in the refinement, each with occupancy 0.5. the final Rfree was 27.7% (2.8A data). Prior to introduction of the second copy of F1, the Rfree was 37%. More details are in Cabezon et al., NSMB 10, 744-750, 2003 Best wishes, Andrew On 11 Mar 2014, at 14:04, Stephen Cusack cus...@embl.fr wrote: Dear All, I have 2.6 A data and unambiguous molecular replacement solution for two copies/asymmetric unit of a 80 K protein for a crystal integrated in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. Refinement allowed rebuilding/completion of the model in the noraml way but the R-free does not go below 30%. The map in the model regions looks generally fine but there is a lot of extra positive density in the solvent regions (some of it looking like weak density for helices and strands) and unexpected positive peaks within the model region. Careful inspection allowed manual positioning of a completely different, overlapping solution for the dimer which fits the extra density perfectly. The two incompatible solutions are related by a 2-fold axis parallel to a. This clearly suggests some kind of twinning. However twinning analysis programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of pseudo-merohedral twinning (-h, l, k) do not reveal any significant twinning fraction and proclaim the data likely to be untwinned. (NB. The programmes do however highlight a non-crystallographic translation and there are systematic intensity differences in the data). Refinement, including this twinning law made no difference since the estimated twinning fraction was 0.02. Yet the extra density is clearly there and I know exactly the real-space transformation between the two packing solutions. How can I best take into account this alternative solution (occupancy seems to be around 20-30%) in the refinement ? thanks for your suggestions Stephen -- ** Dr. Stephen Cusack, Head of Grenoble Outstation of EMBL Group leader in structural biology of protein-RNA complexes and viral proteins Joint appointment in EMBL Genome Biology Programme Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell Interactions (UVHCI) ** Email:cus...@embl.fr Website: http://www.embl.fr Tel: (33) 4 76 20 7238Secretary (33) 4 76 20 7123 Fax:(33) 4 76 20 7199 Postal address: EMBL Grenoble Outstation, 6 Rue Jules Horowitz, BP181, 38042 Grenoble Cedex 9, France Delivery address: EMBL Grenoble Outstation, Polygone Scientifique, 6 Rue Jules Horowitz, 38042 Grenoble, France **
Re: [ccp4bb] twinning problem ?
Not sure I understand why having statistical disorder makes for streaks--does the crystal then have a whole range of unit cell constants, with the spot at the most prevalent value, and the streaks are the tails of the distribution? If so, doesn't having the streak imply a really wide range of constants? And how would this be different from mosaicity? My guess is that this is not the right picture, and this is indeed roughly what mosaicity is. Alternatively, perhaps the streaks are interpreted as the result of a duality between the unit cell, which yields spots, and a super cell which is so large that it yields extremely close spots which are indistinguishable from lines/streaks. Usually this potential super cell is squelched by destructive interference due to each component unit cell being very nearly identical, but here the destructive interference doesn't happen because each component unit cell differs quite a bit from its fellows. And I guess in the latter case the supercell would have its cell constant (in the direction of the streaks) equal to (or a function of) the coherence length of the incident radiation? I know some attempts have been (successfully) made to use diffuse scattering, but has anyone used the streak intensities to determine interesting features of the crystallized protein? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew Leslie Sent: Wednesday, March 12, 2014 12:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] twinning problem ? Dear Stephen, I have seen a similar effect in the structure of F1-ATPase complexed with the full length inhibitor protein. The inhibitor is a dimer, and it actually couples 2 copies of the ATPase, but it crystallised with only one copy of the ATPase per asymmetric unit. When I solved the structure by MR, I saw additional density that could not be accounted for. The extra density was, in fact, a second ATPase molecule that was related to the first by a 120 degree rotation about the pseudo 3-fold axis of the enzyme. The dimers were packing with statistical disorder in the crystal lattice. This gave rise to clear streaking between Bragg spots in the diffraction images in a direction that was consistent with that expected from the statistical packing of the inhibitor linked dimers. Two copies of F1 were included in the refinement, each with occupancy 0.5. the final Rfree was 27.7% (2.8A data). Prior to introduction of the second copy of F1, the Rfree was 37%. More details are in Cabezon et al., NSMB 10, 744-750, 2003 Best wishes, Andrew On 11 Mar 2014, at 14:04, Stephen Cusack cus...@embl.fr wrote: Dear All, I have 2.6 A data and unambiguous molecular replacement solution for two copies/asymmetric unit of a 80 K protein for a crystal integrated in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. Refinement allowed rebuilding/completion of the model in the noraml way but the R-free does not go below 30%. The map in the model regions looks generally fine but there is a lot of extra positive density in the solvent regions (some of it looking like weak density for helices and strands) and unexpected positive peaks within the model region. Careful inspection allowed manual positioning of a completely different, overlapping solution for the dimer which fits the extra density perfectly. The two incompatible solutions are related by a 2-fold axis parallel to a. This clearly suggests some kind of twinning. However twinning analysis programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of pseudo-merohedral twinning (-h, l, k) do not reveal any significant twinning fraction and proclaim the data likely to be untwinned. (NB. The programmes do however highlight a non-crystallographic translation and there are systematic intensity differences in the data). Refinement, including this twinning law made no difference since the estimated twinning fraction was 0.02. Yet the extra density is clearly there and I know exactly the real-space transformation between the two packing solutions. How can I best take into account this alternative solution (occupancy seems to be around 20-30%) in the refinement ? thanks for your suggestions Stephen -- ** Dr. Stephen Cusack, Head of Grenoble Outstation of EMBL Group leader in structural biology of protein-RNA complexes and viral proteins Joint appointment in EMBL Genome Biology Programme Director of CNRS-UJF-EMBL International Unit (UMI 3265) for Virus Host Cell Interactions (UVHCI) ** Email:cus...@embl.fr Website: http://www.embl.fr Tel: (33) 4 76 20 7238Secretary (33) 4 76 20 7123 Fax:(33)
Re: [ccp4bb] twinning problem ?
How to approach the analysis of such a problem: For any sample, crystalline or not, a generally valid description of diffraction intensity is it being a Fourier transform of electron density autocorrelation function. There are obvious normalizations involved. For crystals, this autocorrelation function is periodic and is called a Patterson function when it is derived from diffraction data. In the case of statistical disorder, an important factor characterizing it is autocorrelation of alternative conformations when they are displaced by unit cell periodicities. If such autocorrelation is zero, we have a pure statistical disorder; in such a case, we should add structure factors of alternative conformations to create a calculated F. There will be also diffused scattering from the disorder, but it will not be aligned with Bragg diffraction. More often, the presence of a particular alternative conformation will affect the probability of alternative conformation a unit cell away, and this needs to be considered separately for every unit cell translation. If this correlation is very strong - close to 1 - we have a situation similar or identical to merohedral twinning, and one should add F^2 from alternative models. In an intermediate case, when autocorrelation in a particular direction is between zero and one, the Fourier transform will produce streaks in diffraction pattern and the alignment of these streaks will be related to the properties of the autocorrelation function. Unfortunately, this creates problems when dealing with reduced data sets. Mosaicity is a very different phenomenon. It describes a range of angular alignments of microcrystals with the same unit cell within the sample. It broadens diffraction peaks by the same angle irrespective of the data resolution, but it cannot change the length of diffraction vector for each Bragg reflection. For this reason, the elongation of the spot on the detector resulting from mosaicity will be always perpendicular to the diffraction vector. This is distinct from the statistical disorder, where spot elongation will be aligned with the crystal lattice and not the detector plane. Obviously, no phase information can be derived from the spot shapes resulting from mosaicity. Interestingly, there is a potential for extracting phase information from spot shapes induced by statistical disorder. However, it is far from simple and can be used only to improve phases. It is not promising as an ab initio phasing method. This discussion assumed only one unit cell periodicity in the sample, which is the desired state in all cases. In cryo-cooled crystals, the rate of cooling is different for different parts of the sample, resulting quite often in different unit cell periodicities across the sample. Now there are multiple possibilities to consider; quite typically, the crystal symmetry is the same and the range of unit cell variability is small. This results in variable spot shape elongation, with angular range being resolution-dependent and elongation not necessarily perpendicular to the diffraction vector. By just looking at diffraction pattern, it is easy to distinguish this case from mosaicity. In such samples, a problem arises when rotation exposes distinctly different phases at different orientations. The resulting diffraction data will merge with poor statistics, as distinct structure factors will be merged together. Such condition is quite typical when large crystals are exposed with microbeams. Presence of different crystal forms also provides phasing opportunities known as averaging between crystals. However, this requires separate data set collection rather than mixing such crystals during one rotation sweep. Presence of multiple, similar unit cells in the sample is completely different and unrelated condition to statistical disorder. Zbyszek Otwinowski Not sure I understand why having statistical disorder makes for streaks--does the crystal then have a whole range of unit cell constants, with the spot at the most prevalent value, and the streaks are the tails of the distribution? If so, doesn't having the streak imply a really wide range of constants? And how would this be different from mosaicity? My guess is that this is not the right picture, and this is indeed roughly what mosaicity is. Alternatively, perhaps the streaks are interpreted as the result of a duality between the unit cell, which yields spots, and a super cell which is so large that it yields extremely close spots which are indistinguishable from lines/streaks. Usually this potential super cell is squelched by destructive interference due to each component unit cell being very nearly identical, but here the destructive interference doesn't happen because each component unit cell differs quite a bit from its fellows. And I guess in the latter case the supercell would have its cell constant (in the direction of the streaks) equal to (or a function of) the coherence length
Re: [ccp4bb] twinning problem ?
Dear Jacob For a review of this topic see http://www.tandfonline.com/doi/full/10.1080/08893110310001643551#.UyCVLikgGc0 I also refer you to the more recent OUP IUCr book Chayen, Helliwell and Snell ie which includes these topics:- http://global.oup.com/academic/product/macromolecular-crystallization-and-crystal-perfection-9780199213252;jsessionid=5564F908743CCE57BAD506586B47B6CC?cc=gblang=en; I declare a 'perceived conflict of interest' in making this book suggestion to you. Best wishes John Prof John R Helliwell DSc On 12 Mar 2014, at 16:59, Keller, Jacob kell...@janelia.hhmi.org wrote: Not sure I understand why having statistical disorder makes for streaks--does the crystal then have a whole range of unit cell constants, with the spot at the most prevalent value, and the streaks are the tails of the distribution? If so, doesn't having the streak imply a really wide range of constants? And how would this be different from mosaicity? My guess is that this is not the right picture, and this is indeed roughly what mosaicity is. Alternatively, perhaps the streaks are interpreted as the result of a duality between the unit cell, which yields spots, and a super cell which is so large that it yields extremely close spots which are indistinguishable from lines/streaks. Usually this potential super cell is squelched by destructive interference due to each component unit cell being very nearly identical, but here the destructive interference doesn't happen because each component unit cell differs quite a bit from its fellows. And I guess in the latter case the supercell would have its cell constant (in the direction of the streaks) equal to (or a function of) the coherence length of the incident radiation? I know some attempts have been (successfully) made to use diffuse scattering, but has anyone used the streak intensities to determine interesting features of the crystallized protein? JPK -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Andrew Leslie Sent: Wednesday, March 12, 2014 12:25 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] twinning problem ? Dear Stephen, I have seen a similar effect in the structure of F1-ATPase complexed with the full length inhibitor protein. The inhibitor is a dimer, and it actually couples 2 copies of the ATPase, but it crystallised with only one copy of the ATPase per asymmetric unit. When I solved the structure by MR, I saw additional density that could not be accounted for. The extra density was, in fact, a second ATPase molecule that was related to the first by a 120 degree rotation about the pseudo 3-fold axis of the enzyme. The dimers were packing with statistical disorder in the crystal lattice. This gave rise to clear streaking between Bragg spots in the diffraction images in a direction that was consistent with that expected from the statistical packing of the inhibitor linked dimers. Two copies of F1 were included in the refinement, each with occupancy 0.5. the final Rfree was 27.7% (2.8A data). Prior to introduction of the second copy of F1, the Rfree was 37%. More details are in Cabezon et al., NSMB 10, 744-750, 2003 Best wishes, Andrew On 11 Mar 2014, at 14:04, Stephen Cusack cus...@embl.fr wrote: Dear All, I have 2.6 A data and unambiguous molecular replacement solution for two copies/asymmetric unit of a 80 K protein for a crystal integrated in P212121 (R-merge around 9%) with a=101.8, b=132.2, c=138.9. Refinement allowed rebuilding/completion of the model in the noraml way but the R-free does not go below 30%. The map in the model regions looks generally fine but there is a lot of extra positive density in the solvent regions (some of it looking like weak density for helices and strands) and unexpected positive peaks within the model region. Careful inspection allowed manual positioning of a completely different, overlapping solution for the dimer which fits the extra density perfectly. The two incompatible solutions are related by a 2-fold axis parallel to a. This clearly suggests some kind of twinning. However twinning analysis programmes (e.g. Phenix-Xtriage), while suggesting the potentiality of pseudo-merohedral twinning (-h, l, k) do not reveal any significant twinning fraction and proclaim the data likely to be untwinned. (NB. The programmes do however highlight a non-crystallographic translation and there are systematic intensity differences in the data). Refinement, including this twinning law made no difference since the estimated twinning fraction was 0.02. Yet the extra density is clearly there and I know exactly the real-space transformation between the two packing solutions. How can I best take into account this alternative solution (occupancy seems to be around 20-30%) in the refinement ? thanks for your suggestions
[ccp4bb] Help in getting source of HOLE programme
Hello everyone, I request all of you to please help in getting the source of hole programme which is required to visualize the cavity running through the channel in protein structures. This programme is written by Dr. Oliver Smart. Despite of exhaustive search on various place on web, I am unable to find the source of this programme and therefor request to you, if any one has the source or link to get this progammes please provide me. I will be highly thank full for the help. Thank you Appu
Re: [ccp4bb] Help in getting source of HOLE programme
AFAIK, Dr Smart now works at Global phasing. https://www.globalphasing.com/people/osmart/ HTH, Dave Dr David C Briggs PhD http://about.me/david_briggs On 12 Mar 2014 19:46, Appu kumar appu.kum...@gmail.com wrote: Hello everyone, I request all of you to please help in getting the source of hole programme which is required to visualize the cavity running through the channel in protein structures. This programme is written by Dr. Oliver Smart. Despite of exhaustive search on various place on web, I am unable to find the source of this programme and therefor request to you, if any one has the source or link to get this progammes please provide me. I will be highly thank full for the help. Thank you Appu
[ccp4bb] Refmac bond restraints across special positions?
Colleagues, We have determined a structure of a palindromic DNA molecule, in which one half of the DNA is in the asymmetric unit. Is there a way to tell REFMAC that there are covalent bonds across asymmetric units? Without such LINK records in the PDB file, REFMAC treats this as a non-covalent interaction and pushes the two DNA halfs apart. The data are at a fairly high resolution, which helps, but the repulsion is still there. Any advice would be greatly appreciated! I imagine this situation is quite rare in macromolecular crystallography. Oleg -- Oleg Tsodikov, Ph.D. Associate Professor of Pharmaceutical Sciences University of Kentucky College of Pharmacy Department of Pharmaceutical Sciences BioPharm Bldg, Room 425 789 S. Limestone Lexington, KY 40536
Re: [ccp4bb] Refmac bond restraints across special positions?
I haven’t tried this in a long time, but in the old days, we would have simply refined one strand. On Mar 12, 2014, at 4:05 PM, Oleg Tsodikov olegtsodi...@gmail.com wrote: Colleagues, We have determined a structure of a palindromic DNA molecule, in which one half of the DNA is in the asymmetric unit. Is there a way to tell REFMAC that there are covalent bonds across asymmetric units? Without such LINK records in the PDB file, REFMAC treats this as a non-covalent interaction and pushes the two DNA halfs apart. The data are at a fairly high resolution, which helps, but the repulsion is still there. Any advice would be greatly appreciated! I imagine this situation is quite rare in macromolecular crystallography. Oleg -- Oleg Tsodikov, Ph.D. Associate Professor of Pharmaceutical Sciences University of Kentucky College of Pharmacy Department of Pharmaceutical Sciences BioPharm Bldg, Room 425 789 S. Limestone Lexington, KY 40536
Re: [ccp4bb] twinning problem ?
For any sample, crystalline or not, a generally valid description of diffraction intensity is it being a Fourier transform of electron density autocorrelation function. I thought for non-crystalline samples diffraction intensity is simply the Fourier transform of the electron density, not its autocorrelation function. Is that wrong? Anyway, regarding spot streaking, perhaps there is a different, simpler formulation for how they arise, based on the two phenomena: (1) Crystal lattice convoluted with periodic contents, e.g., protein structure in exactly the same orientation (2) Crystal lattice convoluted with aperiodic contents, e.g. n different conformations of a protein loop, randomly sprinkled in the lattice. Option (1) makes normal spots. If there is a lot of scattering material doing (2), then streaks arise due to many super-cells occurring, each with an integral number of unit cells, and following a Poisson distribution with regard to frequency according to the number of distinct conformations. Anyway, I thought of this because it might be related to scattering from aperiodic crystals, in which there is no concept of unit cell as far as I know (just frequent distances), which makes them really interesting for thinking about diffraction. See the images here of an aperiodic lattice and its Fourier transform, if interested: http://postimg.org/gallery/1fowdm00/ Mosaicity is a very different phenomenon. It describes a range of angular alignments of microcrystals with the same unit cell within the sample. It broadens diffraction peaks by the same angle irrespective of the data resolution, but it cannot change the length of diffraction vector for each Bragg reflection. For this reason, the elongation of the spot on the detector resulting from mosaicity will be always perpendicular to the diffraction vector. This is distinct from the statistical disorder, where spot elongation will be aligned with the crystal lattice and not the detector plane. I have been convinced by some elegant, carefully-thought-out papers that this microcrystal conception of the data-processing constant mosaicity is basically wrong, and that the primary factor responsible for observed mosaicity is discrepancies in unit cell constants, and not the microcrystal picture. I think maybe you are referring here to theoretical mosaicity and not the fitting parameter, so I am not contradicting you. I have seen recently an EM study of protein microcrystals which seems to show actual tilted mosaic domains just as you describe, and can find the reference if desired. Presence of multiple, similar unit cells in the sample is completely different and unrelated condition to statistical disorder. Agreed! Jacob
Re: [ccp4bb] Refmac bond restraints across special positions?
Hi, Is there a way to tell REFMAC that there are covalent bonds across asymmetric units? Try this (example from 3gbi.pdb) for DNA: LINK PDC B 119 O3' DA B 125 1555 2555 1.61 LINK O3' DA B 125 PDC B 119 1555 3555 1.61 LINK PDG C 209 O3' DT D 108 1555 3555 1.61 LINK O3' DT D 108 PDG C 209 1555 2555 1.61 Best, Debanu. -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Craig Bingman Sent: Wednesday, March 12, 2014 2:10 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Refmac bond restraints across special positions? I haven't tried this in a long time, but in the old days, we would have simply refined one strand. On Mar 12, 2014, at 4:05 PM, Oleg Tsodikov olegtsodi...@gmail.com wrote: Colleagues, We have determined a structure of a palindromic DNA molecule, in which one half of the DNA is in the asymmetric unit. Is there a way to tell REFMAC that there are covalent bonds across asymmetric units? Without such LINK records in the PDB file, REFMAC treats this as a non-covalent interaction and pushes the two DNA halfs apart. The data are at a fairly high resolution, which helps, but the repulsion is still there. Any advice would be greatly appreciated! I imagine this situation is quite rare in macromolecular crystallography. Oleg -- Oleg Tsodikov, Ph.D. Associate Professor of Pharmaceutical Sciences University of Kentucky College of Pharmacy Department of Pharmaceutical Sciences BioPharm Bldg, Room 425 789 S. Limestone Lexington, KY 40536
[ccp4bb] Computational postdoctoral position at LBNL
Posted on behalf of Peter Zwart in the Berkeley Center for Structural Biology: Dear All, The Berkeley Center for Structural Biology at Lawrence Berkeley National Lab is looking for a postdoctoral fellow to work on the development of computational methods aimed at improving the overall quality of crystallographic data and its resulting atomic models. The results of this work will be integrated into the Berkeley Center for Structural Biology (BCSB) crystallography beam lines at the Advanced Light Source. Please have a look at the job posting and contact me for more information: https://lbl.taleo.net/careersection/2/jobdetail.ftl?lang=enjob=77796 Regards Peter Zwart -- - P.H. Zwart Research Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org SASTBX: http://sastbx.als.lbl.gov - -- via: Nicholas K. Sauter, Ph. D. Computer Staff Scientist, Physical Biosciences Division Lawrence Berkeley National Laboratory 1 Cyclotron Rd., Bldg. 64R0121 Berkeley, CA 94720-8118 (510) 486-5713
Re: [ccp4bb] twinning problem ?
On 03/12/2014 04:15 PM, Keller, Jacob wrote: For any sample, crystalline or not, a generally valid description of diffraction intensity is it being a Fourier transform of electron density autocorrelation function. I thought for non-crystalline samples diffraction intensity is simply the Fourier transform of the electron density, not its autocorrelation function. Is that wrong? The Fourier transform of electron density is a complex scattering amplitude that by the axiom of quantum mechanics is not a measurable quantity. What is measurable is the module squared of it. In crystallography, it is called either F^2 (formally equal F*Fbar) or somewhat informally diffraction intensity, after one takes into account scaling factors. F*Fbar is the Fourier transform of an electron density autocorrelation function regardless if electron density is periodic or not. For periodic electron density the structure factors are described by sum of delta Dirac functions placed on the reciprocal lattice. These delta functions are multiplied by values of structure factors for corresponding Miller indices. Anyway, regarding spot streaking, perhaps there is a different, simpler formulation for how they arise, based on the two phenomena: (1) Crystal lattice convoluted with periodic contents, e.g., protein structure in exactly the same orientation (2) Crystal lattice convoluted with aperiodic contents, e.g. n different conformations of a protein loop, randomly sprinkled in the lattice. Option (1) makes normal spots. If there is a lot of scattering material doing (2), then streaks arise due to many super-cells occurring, each with an integral number of unit cells, and following a Poisson distribution with regard to frequency according to the number of distinct conformations. Anyway, I thought of this because it might be related to scattering from aperiodic crystals, in which there is no concept of unit cell as far as I know (just frequent distances), which makes them really interesting for thinking about diffraction. This formulation cannot describe aperiodic contents. The convolution of a crystal lattice with any function will result in electron density, which has a perfect crystal symmetry of the same periodicity as the starting crystal lattice. See the images here of an aperiodic lattice and its Fourier transform, if interested: http://postimg.org/gallery/1fowdm00/ This is interesting case of pseudocrystal, however because there is no crystal lattice, it is not relevant to (1) or (2). In any case, pentagonal quasilattices are probably not relevant to macromolecular crystallography. Mosaicity is a very different phenomenon. It describes a range of angular alignments of microcrystals with the same unit cell within the sample. It broadens diffraction peaks by the same angle irrespective of the data resolution, but it cannot change the length of diffraction vector for each Bragg reflection. For this reason, the elongation of the spot on the detector resulting from mosaicity will be always perpendicular to the diffraction vector. This is distinct from the statistical disorder, where spot elongation will be aligned with the crystal lattice and not the detector plane. I have been convinced by some elegant, carefully-thought-out papers that this microcrystal conception of the data-processing constant mosaicity is basically wrong, and that the primary factor responsible for observed mosaicity is discrepancies in unit cell constants, and not the microcrystal picture. I think maybe you are referring here to theoretical mosaicity and not the fitting parameter, so I am not contradicting you. I have seen recently an EM study of protein microcrystals which seems to show actual tilted mosaic domains just as you describe, and can find the reference if desired. This is easy to test by analyzing diffraction patterns of individual crystals. In practice, the dominant contribution to angular broadening of diffraction peaks is angular disorder of microdomains, particularly in cryo-cooled crystals. However, exceptions do happen, but these rare situations need to be handled on case by case basis. Zbyszek Presence of multiple, similar unit cells in the sample is completely different and unrelated condition to statistical disorder. Agreed! Jacob -- Zbyszek Otwinowski UT Southwestern Medical Center 5323 Harry Hines Blvd., Dallas, TX 75390-8816 (214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu
Re: [ccp4bb] twinning problem ?
The Fourier transform of electron density is a complex scattering amplitude that by the axiom of quantum mechanics is not a measurable quantity. What is measurable is the module squared of it. In crystallography, it is called either F^2 (formally equal F*Fbar) or somewhat informally diffraction intensity, after one takes into account scaling factors. F*Fbar is the Fourier transform of an electron density autocorrelation function regardless if electron density is periodic or not. For periodic electron density the structure factors are described by sum of delta Dirac functions placed on the reciprocal lattice. These delta functions are multiplied by values of structure factors for corresponding Miller indices. Okay, I may have been confused--I thought that the Fourier transform was essentially acting like an autocorrelation function (since generally Fourier transforms are similar to autocorrelation functions--not clear on the details right now), and I had thought I had heard stories of days of yore handwritten Fourier series calculations to make electron density maps. You're telling me they had to also back-calculate an autocorrelation function? Times were tough! Maybe someone from that generation can chime in about how they dealt with this? This is interesting case of pseudocrystal, however because there is no crystal lattice, it is not relevant to (1) or (2). In any case, pentagonal quasilattices are probably not relevant to macromolecular crystallography. I tried a few simulations to show what I mean but ran out of time--sorry about that. I think I'll probably just drop this. NB Linus Pauling said more forcefully the same prediction about aperiodic crystals in general not existing, pentagonal or otherwise, but was proven dead wrong by now-Nobel laureate Dan Shechtman. Maybe someone will come across an aperiodic protein crystal, or already has and missed it, and stupefy us all. Someone mentioned to me once seeing personally a ten-fold symmetrical diffraction pattern from a protein crystal, but she dismissed it with exactly the argument that Pauling made, I think that it was a twinned cubic space group. This is easy to test by analyzing diffraction patterns of individual crystals. In practice, the dominant contribution to angular broadening of diffraction peaks is angular disorder of microdomains, particularly in cryo-cooled crystals. However, exceptions do happen, but these rare situations need to be handled on case by case basis. I was thinking of this paper for example (see last line of abstract). Perhaps other crystals are different from lysozyme, though, as you mention. All the best, Jacob Keller Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):848-53. A description of imperfections in protein crystals. Nave C. Author information Abstract An analysis is given of the contribution of various crystal imperfections to the rocking widths of reflections and the divergence of the diffracted beams. The crystal imperfections are the angular spread of the mosaic blocks in the crystal, the size of the mosaic blocks and the variation in cell dimensions between blocks. The analysis has implications for improving crystal perfection, defining data-collection requirements and for data-processing procedures. Measurements on crystals of tetragonal lysozyme at room temperature and 100 K were made in order to illustrate how parameters describing the crystal imperfections can be obtained. At 100 K, the dominant imperfection appeared to be a variation in unit-cell dimensions in the crystal. PMID: 9757100 [PubMed - indexed for MEDLINE]
Re: [ccp4bb] twinning problem ?
On 03/12/2014 09:02 PM, Keller, Jacob wrote: The Fourier transform of electron density is a complex scattering amplitude that by the axiom of quantum mechanics is not a measurable quantity. What is measurable is the module squared of it. In crystallography, it is called either F^2 (formally equal F*Fbar) or somewhat informally diffraction intensity, after one takes into account scaling factors. F*Fbar is the Fourier transform of an electron density autocorrelation function regardless if electron density is periodic or not. For periodic electron density the structure factors are described by sum of delta Dirac functions placed on the reciprocal lattice. These delta functions are multiplied by values of structure factors for corresponding Miller indices. Okay, I may have been confused--I thought that the Fourier transform was essentially acting like an autocorrelation function (since generally Fourier transforms are similar to autocorrelation functions--not clear on the details right now), and I had thought I had heard stories of days of yore handwritten Fourier series calculations to make electron density maps. You're telling me they had to also back-calculate an autocorrelation function? Times were tough! Maybe someone from that generation can chime in about how they dealt with this? Even in today’s easy times, the fastest way to calculate autocorrelation function is to calculate Fourier transform of the data, calculate F*Fbar and calculate back Fourier transform of it. This is interesting case of pseudocrystal, however because there is no crystal lattice, it is not relevant to (1) or (2). In any case, pentagonal quasilattices are probably not relevant to macromolecular crystallography. I tried a few simulations to show what I mean but ran out of time--sorry about that. I think I'll probably just drop this. NB Linus Pauling said more forcefully the same prediction about aperiodic crystals in general not existing, pentagonal or otherwise, but was proven dead wrong by now-Nobel laureate Dan Shechtman. Maybe someone will come across an aperiodic protein crystal, or already has and missed it, and stupefy us all. Someone mentioned to me once seeing personally a ten-fold symmetrical diffraction pattern from a protein crystal, but she dismissed it with exactly the argument that Pauling made, I think that it was a twinned cubic space group. Unless you are interested in finding curious objects, what would you do with protein quasicrystal? The practices of macromolecular crystallography is about determining 3-dimensional structure of objects being crystallized. Protein quasicrystal are really unlikely to diffract to high enough resolution, and even ignoring all other practical aspects, like writing programs to solve such a structure, chances of building an atomic model are really slim. This is easy to test by analyzing diffraction patterns of individual crystals. In practice, the dominant contribution to angular broadening of diffraction peaks is angular disorder of microdomains, particularly in cryo-cooled crystals. However, exceptions do happen, but these rare situations need to be handled on case by case basis. The interpretation of the data presented in this article is that variation in unit cell between microcrystals induce their spatial misalignment. The data do not show variation of unit cell within individual microscrystalline domains. Tetragonal lysozyme can adopt quite a few variations of the crystal lattice during cryocooling. Depending on the conditions used, resulting mosaicity can vary from 0.1 degree (even for 1mm size crystal) to over 1. degree. Consequently, measured structure factors from a group of tetragonal lysozyme crystal can be quite reproducible, or not. As a test crystal, it should be handled with care. 1 degree mosaicity is not an impediment to high quality measurements. However, high mosaicity tends to correlate with presence of phase transitions during cryo-cooling. If such transition happen during cryo-cooling, crystals of the same protein, even from the same drop, may vary quite a lot in terms of structure factors. Additionally, even similar values of unit cell parameters are not guarantee of isomorphism between crystals. Zbyszek I was thinking of this paper for example (see last line of abstract). Perhaps other crystals are different from lysozyme, though, as you mention. All the best, Jacob Keller Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):848-53. A description of imperfections in protein crystals. Nave C. Author information Abstract An analysis is given of the contribution of various crystal imperfections to the rocking widths of reflections and the divergence of the diffracted beams. The crystal imperfections are the angular spread of the mosaic blocks in the crystal, the size of the mosaic blocks and the variation in cell dimensions between blocks. The analysis has implications for
