Re: [ccp4bb] Invisible atoms in ligands
Thank you Pavel for the clarification! What I was really trying to point out is that a missing atom, occ=0.00 and occ=0.01 are not as similar as one might naiively think. Also, if you put a ligand into a pocket and the occupancy refines to 0, that does not necessarily mean the ligand is partially occupied. If the pocket is actually filled with flat bulk solvent, then you expect the ligand occupancy to be non-zero, simply because something is better than nothing. However, if the bulk solvent mask were somehow smarter and filled the pocket of a, say, 60% occupied ligand with flat bulk density at 40% the level of bulk density used far away from any atoms, then one might actually see the occupancy of a bogus ligand refine to zero. That is, a ligand built into a pocket that is truly empty (filled with flat bulk solvent) and then occupancy refined would actually be a competition between two alternative hypotheses: 1) ligand in the pocket, 2) nothing but solvent in the pocket. If the occupancy of the ligand refines to zero in this context, then you can be quite confident that it didn't bind, at least not in the given orientation. I fully realize that the implementation of this is easier said than done, but perhaps it would be worth the effort? -James Holton MAD Scientist On 6/16/2014 3:04 PM, Pavel Afonine wrote: Hi James, a remark: different programs may treat occ=0 differently. In phenix.refine (phenix.maps, etc) atoms with zero occupancy will be ignored for bulk-solvent mask calculation, unless you ask to do otherwise. For example, this means that if you want to calculate a ligand OMIT map both options - removing the ligand from PDB file; - setting its occupancy to zero and making sure mask does not ignore occ=0 atoms) are a) not equivalent and b) both not good. In first case (removing atoms from file) bulk-solvent will flatten residual map (as you pointed out). In second case bulk-solvent will be excluded in a very specific area, so that residual (green) density you see there may be either just bulk-solvent or ligand in question or a mixture; obviously not a very useful information! This highlights the fundamental problem of flat bulk-solvent model the way it's currently used. Pavel On Sun, Jun 15, 2014 at 3:01 PM, James Holton jmhol...@lbl.gov mailto:jmhol...@lbl.gov wrote: The principle difference between occ=0 and omitting the atom entirely is that occ=0 atoms exclude bulk solvent. Or at least they do for typical operation of contemporary refinement programs. So, by defining occ=0 you are forcing all map voxels within ~0.6A or so of your invisible atom to be vacuum. If you omit it, then the bulk solvent may flood in, perhaps enough to pull the fo-fc peak down below 3x rms. How much the bulk solvent floods in depends on how nearby atoms exclude the bulk solvent, and this, in turn, depends on which refinement program you are using. Different bulk solvent implementations use different radii, shrink parameters, etc. In addition, bulk solvent always bleeds a bit into surrounding areas because the solvent B factor is never zero. The real problem, I think, is that for any voxel of the map there is ALWAYS something there. The only question is: what is it? Is there a 100% occupied ligand? 100% occupied solvent? Two conformers of the ligand? Or is it some mixture of all these? If you are asking these questions I think it is most likely a mixture, and mixtures are hard to model. What is worse, mixtures of a partially-occupied ligand with bulk solvent taking up the slack is currently impossible to model. We will have to wait for partial-occupancy-bulk-solvent to be implemented before we can build representations of these alternative hypotheses and and test them with competitive occupancy refinement. The bulk solvent is actually a very good example of something for which we see no evidence in our electron density maps, yet we model it in because 1) we know it must be there, and 2) it makes our R factors lower. What more could you want? -James Holton MAD Scientist On 6/13/2014 7:45 PM, Frank von Delft wrote: Hi all - talking about ligands, a quick question on that old conundrum, of what to do about invisible atoms -- build them with occ=0, or omit them? For bits of protein, I know all the arguments; personally I prefer omitting atoms because: * for amino acid sidechains, their presence is implied in the residue name. * for whole residues, their presence is implied in the sequence numbering However: what about ligands? Nowhere else in the PDB file is their presence implied - or have I missed something? (Certainly disorder in a ligand is important information that needs to be captured!) Cheers phx
[ccp4bb] Arp/warp error
Dear all I am getting this error while running arp/warp to build DNA/RNA: PHIB is not assigned to an mtz label Input was merged data (.mtz) However no such problem while using arp/warp classic as i chose for automated model building from existing model and not experimental phases. Kindly help how to give PHIB and FOM labels here?? Thanx in advance Monica
[ccp4bb] Two years post-doc position to work on the structural characterization of protein involved in chronic pain (Scuola Normale Superiore - Pisa, Italy and Institute of Crystallography at CNR, Tri
Dear Structural Biology community, I would be grateful if you could please bring the following postdoctoral position to the attention of potential candidates. Kindest regards Much obliged Doriano Lamba Two years post-doc position to work on the structural characterization of protein involved in chronic pain (Scuola Normale Superiore - Pisa, Italy and Institute of Crystallography at CNR, Trieste, Italy) Research contract: “Dissecting NGF Interactions with TrkA Receptor: Structural Studies of complexes with two Neutralizing Antibodies of therapeutic relevance in chronic pain” on the following European project: The NGF system and its interplay with endocannabinoid signalling, from peripheral sensory terminals to the brain: new targets for the development of next generation drugs for neuropathic pain” We are seeking a highly motivated and enthusiastic post-doc to perform structural studies on molecules of the Nerve Growth Factor (NGF) receptor family and on their antibodies. The position will be in the group of Antonino Cattaneo at Scuola Normale Superiore (Pisa) and of Doriano Lamba at the Institute of Crystallography in Trieste. The Joint groups have a considerable experience in the structural characterization of proteins relevant for neurodegeneration and chronic pain. The experimental work will be performed mostly in Trieste. In this EU funded three-years project (PAINCAGE) the SNS/CNR team will perform the biophysical and structural characterization of complexes between the antiTrkA MNAC13 antibodies in Fab format and the two immunoglobulin-like domains d4-d5 of TrkA and between the antiNGF αD11 antibodies in Fab format and NGF. Moreover the team will perform the structural characterization of the NGFR100 HSAN V mutant. The ideal candidate will have strong skills in molecular biology, protein expression, biochemistry and biophysics/structural biology. Experience in X-ray protein crystallography and/or Small Angle X-ray Scattering are a plus. The candidate should be highly motivated, work well as a team, have excellent communication skills, and a strong publication record. Duration of the contract: 2 years (estimated starting: July/August 2014) Gross compensation inclusive of all taxes: € 47.630,00 Application deadline:July 2nd 2014 To apply please visit: Italian: http://www.sns.it/servizi/job/assegnidiricerca/assegno254/ English: http://www.sns.it/en/servizi/job/assegnidiricerca/assegno254/ For further information please contact: Dr. Doriano Lamba Phone +39 040 375 8514 E-mail: doriano.la...@ts.ic.cnr.it
Re: [ccp4bb] Invisible atoms in ligands
Hi All, I had an interesting case recently where a Cl atom of a chlorophenyl moiety went missing in a structure (primary X-ray damage was the suspected culprit). Small molecule Mass Spec suggested the atom was there to start with but it was quite obviously missing in the maps (1.9Ang) and the des-chloro refined much better. I was asked to replace the missing atom as it was seen as misleading because all the assay and biophysical data was generated with the chlorophenyl compound; this after all, is what the X-ray model was supporting. I did wonder where the Cl atom ended up and in what state. David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.commailto:name.surn...@astrazeneca.com Please consider the environment before printing this e-mail From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft Sent: 13 June 2014 10:45 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Invisible atoms in ligands Hi all - talking about ligands, a quick question on that old conundrum, of what to do about invisible atoms -- build them with occ=0, or omit them? For bits of protein, I know all the arguments; personally I prefer omitting atoms because: * for amino acid sidechains, their presence is implied in the residue name. * for whole residues, their presence is implied in the sequence numbering However: what about ligands? Nowhere else in the PDB file is their presence implied - or have I missed something? (Certainly disorder in a ligand is important information that needs to be captured!) Cheers phx -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies.
[ccp4bb] Help in Cell content analysis
Dear all i have a small query to ask and seek your suggestions: I have collected a data for a protein with 324 residues and processed at its best in P212121. So Matthews suggest 1 mol in ASU with expected Mol. weight of 43 kDa with sovent content of 58% and 2 mol./ASU with 18% solvent content. However the data suggest possibility of translational NCS so i think i should ask for two molecules so that both get corrected for NCS. However for 2 mol./ASU, Matthewssuggests a total mol. weight of 52 kDa. So how to decide which way to proceed for MR? Thanks Monica
Re: [ccp4bb] Arp/warp error
Dear Monica, On Jun 16, 2014, at 10:39 AM, Monica Mittal wrote: Dear all I am getting this error while running arp/warp to build DNA/RNA: PHIB is not assigned to an mtz label Input was merged data (.mtz) However no such problem while using arp/warp classic as i chose for automated model building from existing model and not experimental phases. Kindly help how to give PHIB and FOM labels here?? When you use the ARP/wARP interface and load/browse your .MTZ file, the labels Fobs, PHIB, Sigma, FOM are automatically detected and placed. If you want to select different ones available in your MTZ file, please use the corresponding dropdown menu. First make sure that your MTZ file is correct and contains these labels. You can check that by using mtzdmp command for example, which should be available in your system because is part of CCP4. Best regards, Ioan -- Dr. Ioan Vancea European Molecular Biology Laboratory c/o DESY Notkestrasse 85, 22607 Hamburg, Germany Email: ivan...@embl-hamburg.de
Re: [ccp4bb] Invisible atoms in ligands
Dear James You seem to be discounting the possibility of a true vacuum inside a structure, which is obviously not the same thing as 'something' (bulk solvent or whatever). I accept that this is unlikely in the case of ligand binding sites exposed to solvent, or indeed any site on the outer surface of the molecule, since any vacuum in that situation would be unstable against the ingress of water molecules, but it is possible in the case of fully-enclosed cavities (i.e. 'inner surface') that are normally inaccessible to water. I don't know if anyone has done a systematic survey of this, i.e. looking for cavities where the density appears to be actually zero (taking into account F000 of course), or at least significantly lower compared with the bulk solvent density (where the assumed value of F000 wouldn't affect the result). Cheers -- Ian On 16 June 2014 07:37, James Holton jmhol...@lbl.gov wrote: Thank you Pavel for the clarification! What I was really trying to point out is that a missing atom, occ=0.00 and occ=0.01 are not as similar as one might naiively think. Also, if you put a ligand into a pocket and the occupancy refines to 0, that does not necessarily mean the ligand is partially occupied. If the pocket is actually filled with flat bulk solvent, then you expect the ligand occupancy to be non-zero, simply because something is better than nothing. However, if the bulk solvent mask were somehow smarter and filled the pocket of a, say, 60% occupied ligand with flat bulk density at 40% the level of bulk density used far away from any atoms, then one might actually see the occupancy of a bogus ligand refine to zero. That is, a ligand built into a pocket that is truly empty (filled with flat bulk solvent) and then occupancy refined would actually be a competition between two alternative hypotheses: 1) ligand in the pocket, 2) nothing but solvent in the pocket. If the occupancy of the ligand refines to zero in this context, then you can be quite confident that it didn't bind, at least not in the given orientation. I fully realize that the implementation of this is easier said than done, but perhaps it would be worth the effort? -James Holton MAD Scientist On 6/16/2014 3:04 PM, Pavel Afonine wrote: Hi James, a remark: different programs may treat occ=0 differently. In phenix.refine (phenix.maps, etc) atoms with zero occupancy will be ignored for bulk-solvent mask calculation, unless you ask to do otherwise. For example, this means that if you want to calculate a ligand OMIT map both options - removing the ligand from PDB file; - setting its occupancy to zero and making sure mask does not ignore occ=0 atoms) are a) not equivalent and b) both not good. In first case (removing atoms from file) bulk-solvent will flatten residual map (as you pointed out). In second case bulk-solvent will be excluded in a very specific area, so that residual (green) density you see there may be either just bulk-solvent or ligand in question or a mixture; obviously not a very useful information! This highlights the fundamental problem of flat bulk-solvent model the way it's currently used. Pavel On Sun, Jun 15, 2014 at 3:01 PM, James Holton jmhol...@lbl.gov wrote: The principle difference between occ=0 and omitting the atom entirely is that occ=0 atoms exclude bulk solvent. Or at least they do for typical operation of contemporary refinement programs. So, by defining occ=0 you are forcing all map voxels within ~0.6A or so of your invisible atom to be vacuum. If you omit it, then the bulk solvent may flood in, perhaps enough to pull the fo-fc peak down below 3x rms. How much the bulk solvent floods in depends on how nearby atoms exclude the bulk solvent, and this, in turn, depends on which refinement program you are using. Different bulk solvent implementations use different radii, shrink parameters, etc. In addition, bulk solvent always bleeds a bit into surrounding areas because the solvent B factor is never zero. The real problem, I think, is that for any voxel of the map there is ALWAYS something there. The only question is: what is it? Is there a 100% occupied ligand? 100% occupied solvent? Two conformers of the ligand? Or is it some mixture of all these? If you are asking these questions I think it is most likely a mixture, and mixtures are hard to model. What is worse, mixtures of a partially-occupied ligand with bulk solvent taking up the slack is currently impossible to model. We will have to wait for partial-occupancy-bulk-solvent to be implemented before we can build representations of these alternative hypotheses and and test them with competitive occupancy refinement. The bulk solvent is actually a very good example of something for which we see no evidence in our electron density maps, yet we model it in because 1) we know it must be there, and 2) it makes our R factors lower. What more could you want?
Re: [ccp4bb] Help in Cell content analysis
That is puzzling. 18% solvent is not common and you would expect very strong diffraction with such a low solvent content. one possibility is that the NC symmetry is parallel to a crystal axis and is making monoclinic data appear to have an extra 2-fold axis. (There is a case I have heard of wherethe SG turned out to be P21 and not P2i2i2i - done by Michael Isupov. You could probably find a reference to it) I would look carefully at the pointless summary of the quality of the symmetry operators. If one 2-fold is better than the other two maybe process the data with that 2 fold only.. Of course another possibility is that the protein has been chewed up in crystallisation! Do you have an MR model? Eleanor
[ccp4bb] Kabat, insertion codes refinement
Dear CCP4bb, I'm refining an antibody structure which requires Kabat residue numbering with insertion codes. My setup of Refmac5 and Buster both break peptide bonds between some (not all) of the residues with insertion codes. I was wondering whether there is a special way of handling these residues in refinement? Thanks, David David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.commailto:name.surn...@astrazeneca.com Please consider the environment before printing this e-mail -- AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking Compliance with our Code of Conduct and Policies.
Re: [ccp4bb] Invisible atoms in ligands
Dear Ian, This has been discussed in a review and related articles by Brian Matthews and Liljun Liu: Matthews BW, Liu L. A review about nothing: are apolar cavities in proteins really empty? Protein Sci. 2009 Mar;18(3):494-502. doi: 10.1002/pro.61. Review. PubMed PMID: 19241368; PubMed Central PMCID: PMC2760356. Daniel Le 16/06/2014 11:32, Ian Tickle a écrit : Dear James You seem to be discounting the possibility of a true vacuum inside a structure, which is obviously not the same thing as 'something' (bulk solvent or whatever). I accept that this is unlikely in the case of ligand binding sites exposed to solvent, or indeed any site on the outer surface of the molecule, since any vacuum in that situation would be unstable against the ingress of water molecules, but it is possible in the case of fully-enclosed cavities (i.e. 'inner surface') that are normally inaccessible to water. I don't know if anyone has done a systematic survey of this, i.e. looking for cavities where the density appears to be actually zero (taking into account F000 of course), or at least significantly lower compared with the bulk solvent density (where the assumed value of F000 wouldn't affect the result). Cheers -- Ian On 16 June 2014 07:37, James Holton jmhol...@lbl.gov mailto:jmhol...@lbl.gov wrote: Thank you Pavel for the clarification! What I was really trying to point out is that a missing atom, occ=0.00 and occ=0.01 are not as similar as one might naiively think. Also, if you put a ligand into a pocket and the occupancy refines to 0, that does not necessarily mean the ligand is partially occupied. If the pocket is actually filled with flat bulk solvent, then you expect the ligand occupancy to be non-zero, simply because something is better than nothing. However, if the bulk solvent mask were somehow smarter and filled the pocket of a, say, 60% occupied ligand with flat bulk density at 40% the level of bulk density used far away from any atoms, then one might actually see the occupancy of a bogus ligand refine to zero. That is, a ligand built into a pocket that is truly empty (filled with flat bulk solvent) and then occupancy refined would actually be a competition between two alternative hypotheses: 1) ligand in the pocket, 2) nothing but solvent in the pocket. If the occupancy of the ligand refines to zero in this context, then you can be quite confident that it didn't bind, at least not in the given orientation. I fully realize that the implementation of this is easier said than done, but perhaps it would be worth the effort? -James Holton MAD Scientist On 6/16/2014 3:04 PM, Pavel Afonine wrote: Hi James, a remark: different programs may treat occ=0 differently. In phenix.refine (phenix.maps, etc) atoms with zero occupancy will be ignored for bulk-solvent mask calculation, unless you ask to do otherwise. For example, this means that if you want to calculate a ligand OMIT map both options - removing the ligand from PDB file; - setting its occupancy to zero and making sure mask does not ignore occ=0 atoms) are a) not equivalent and b) both not good. In first case (removing atoms from file) bulk-solvent will flatten residual map (as you pointed out). In second case bulk-solvent will be excluded in a very specific area, so that residual (green) density you see there may be either just bulk-solvent or ligand in question or a mixture; obviously not a very useful information! This highlights the fundamental problem of flat bulk-solvent model the way it's currently used. Pavel On Sun, Jun 15, 2014 at 3:01 PM, James Holton jmhol...@lbl.gov mailto:jmhol...@lbl.gov wrote: The principle difference between occ=0 and omitting the atom entirely is that occ=0 atoms exclude bulk solvent. Or at least they do for typical operation of contemporary refinement programs. So, by defining occ=0 you are forcing all map voxels within ~0.6A or so of your invisible atom to be vacuum. If you omit it, then the bulk solvent may flood in, perhaps enough to pull the fo-fc peak down below 3x rms. How much the bulk solvent floods in depends on how nearby atoms exclude the bulk solvent, and this, in turn, depends on which refinement program you are using. Different bulk solvent implementations use different radii, shrink parameters, etc. In addition, bulk solvent always bleeds a bit into surrounding areas because the solvent B factor is never zero. The real problem, I think, is that for any voxel of the map there is ALWAYS something there. The only question is: what is it? Is there a 100% occupied ligand? 100% occupied solvent? Two conformers of the ligand? Or is it some mixture
Re: [ccp4bb] Invisible atoms in ligands
Dear Daniel Thanks for the info. I knew that Brian Matthews' group had done some work in this area. Cheers -- Ian On 16 June 2014 11:16, Daniel Picot daniel.pi...@ibpc.fr wrote: Dear Ian, This has been discussed in a review and related articles by Brian Matthews and Liljun Liu: Matthews BW, Liu L. A review about nothing: are apolar cavities in proteins really empty? Protein Sci. 2009 Mar;18(3):494-502. doi: 10.1002/pro.61. Review. PubMed PMID: 19241368; PubMed Central PMCID: PMC2760356. Daniel Le 16/06/2014 11:32, Ian Tickle a écrit : Dear James You seem to be discounting the possibility of a true vacuum inside a structure, which is obviously not the same thing as 'something' (bulk solvent or whatever). I accept that this is unlikely in the case of ligand binding sites exposed to solvent, or indeed any site on the outer surface of the molecule, since any vacuum in that situation would be unstable against the ingress of water molecules, but it is possible in the case of fully-enclosed cavities (i.e. 'inner surface') that are normally inaccessible to water. I don't know if anyone has done a systematic survey of this, i.e. looking for cavities where the density appears to be actually zero (taking into account F000 of course), or at least significantly lower compared with the bulk solvent density (where the assumed value of F000 wouldn't affect the result). Cheers -- Ian On 16 June 2014 07:37, James Holton jmhol...@lbl.gov wrote: Thank you Pavel for the clarification! What I was really trying to point out is that a missing atom, occ=0.00 and occ=0.01 are not as similar as one might naiively think. Also, if you put a ligand into a pocket and the occupancy refines to 0, that does not necessarily mean the ligand is partially occupied. If the pocket is actually filled with flat bulk solvent, then you expect the ligand occupancy to be non-zero, simply because something is better than nothing. However, if the bulk solvent mask were somehow smarter and filled the pocket of a, say, 60% occupied ligand with flat bulk density at 40% the level of bulk density used far away from any atoms, then one might actually see the occupancy of a bogus ligand refine to zero. That is, a ligand built into a pocket that is truly empty (filled with flat bulk solvent) and then occupancy refined would actually be a competition between two alternative hypotheses: 1) ligand in the pocket, 2) nothing but solvent in the pocket. If the occupancy of the ligand refines to zero in this context, then you can be quite confident that it didn't bind, at least not in the given orientation. I fully realize that the implementation of this is easier said than done, but perhaps it would be worth the effort? -James Holton MAD Scientist On 6/16/2014 3:04 PM, Pavel Afonine wrote: Hi James, a remark: different programs may treat occ=0 differently. In phenix.refine (phenix.maps, etc) atoms with zero occupancy will be ignored for bulk-solvent mask calculation, unless you ask to do otherwise. For example, this means that if you want to calculate a ligand OMIT map both options - removing the ligand from PDB file; - setting its occupancy to zero and making sure mask does not ignore occ=0 atoms) are a) not equivalent and b) both not good. In first case (removing atoms from file) bulk-solvent will flatten residual map (as you pointed out). In second case bulk-solvent will be excluded in a very specific area, so that residual (green) density you see there may be either just bulk-solvent or ligand in question or a mixture; obviously not a very useful information! This highlights the fundamental problem of flat bulk-solvent model the way it's currently used. Pavel On Sun, Jun 15, 2014 at 3:01 PM, James Holton jmhol...@lbl.gov wrote: The principle difference between occ=0 and omitting the atom entirely is that occ=0 atoms exclude bulk solvent. Or at least they do for typical operation of contemporary refinement programs. So, by defining occ=0 you are forcing all map voxels within ~0.6A or so of your invisible atom to be vacuum. If you omit it, then the bulk solvent may flood in, perhaps enough to pull the fo-fc peak down below 3x rms. How much the bulk solvent floods in depends on how nearby atoms exclude the bulk solvent, and this, in turn, depends on which refinement program you are using. Different bulk solvent implementations use different radii, shrink parameters, etc. In addition, bulk solvent always bleeds a bit into surrounding areas because the solvent B factor is never zero. The real problem, I think, is that for any voxel of the map there is ALWAYS something there. The only question is: what is it? Is there a 100% occupied ligand? 100% occupied solvent? Two conformers of the ligand? Or is it some mixture of all these? If you are asking these questions I think it is most likely a mixture, and mixtures are hard to
Re: [ccp4bb] Hosed-Up X-Ray Structures: A Big Problem
Dear Jeff, I would assume that clashing hydrogen atoms beome less and less an issue with current refinement programs, since those I am familiar with (refmac5 and phenix) both genereate constrained hydrogen atoms by default now, and it has been like this for quite some time - so the situation should become better for modellers. You may also want to note that constrained hydrogen positions are a crude approximation and only work with X-ray data where hydrogen atoms have little impact on the data. Our comparison between hydrogen restraints and constraints (http://dx.doi.org/10.1107/S1600576713027659) report the greater quality of restraints vs. constraints when it comes to neutron data, where hydrogen atoms do matter. Hydrogen positions are much more flexible than the usual riding atom model may imply. This may affect in silico simulations. Best, Tim On 06/16/2014 04:34 AM, Jeffrey Bell wrote: Hi, all, I am glad to see these matters being discussed. I think we all believe that protein crystallographers should be concerned with producing models that modelers and chemists can respect and use. Schrödinger spends a lot of time thinking about ligands; its refinement program, PrimeX, has a very simple way of handling ligand issues. All that a crystallographer has to do is get the charge and bond order right, and the force field then automatically does atom typing and generates all restraints. However, use of our cif library files brings up another matter that must be understood first. Use of PrimeX, even at low resolution, involves refinement of all hydrogen atom positions. The Richardsons have abundantly demonstrated how important hydrogen coordinates are to accurate model building. This matter of hydrogen coordinate refinement is closely connected to the editorial that started this thread. Computational chemistry in drug discovery, and elsewhere, uses all-atom models. When you add hydrogen atoms to most models in the PDB, many chemically-impossible overlaps of atoms result (see Acta Cryst. 2012, D68, 935-952 for more information; ask me for a copy). This issue is almost as much of an annoyance for computational chemists as bad ligand geometry because they see it in almost every structure. If anyone would like to try PrimeX, either for ligand restraint generation or refinement, please let me know. Schrödinger offers academic institutions one year of free access, which may be renewed on a case-by-case basis. Crystallographers at companies will also qualify for a free evaluation trial. Cheers, Jeff Bell PrimeX developer Schrödinger, Inc. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
Re: [ccp4bb] Kabat, insertion codes refinement
There is no actual requirement to use Kabat numbering, you can avoid it alrogether. Some argue that L27A is actually 28th amino acid in the protein sequence, and labeling it as L27A is simply incorrect. I would suggest doing refinement with plain numbering (no insertion codes) and changing it only for the final model if needed for comparative analysis. Ed Sent on a Sprint Samsung Galaxy S® III div Original message /divdivFrom: Hargreaves, David david.hargrea...@astrazeneca.com /divdivDate:06/16/2014 6:07 AM (GMT-05:00) /divdivTo: CCP4BB@JISCMAIL.AC.UK /divdivSubject: [ccp4bb] Kabat, insertion codes refinement /divdiv /divDear CCP4bb, I’m refining an antibody structure which requires Kabat residue numbering with insertion codes. My setup of Refmac5 and Buster both break peptide bonds between some (not all) of the residues with insertion codes. I was wondering whether there is a special way of handling these residues in refinement? Thanks, David David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
Re: [ccp4bb] Help in Cell content analysis
If your translational NCS is defined by a vector that does not correspond to lattice centering, i.e. has numbers different from 0 or 0.5, this is likely a case of order-disorder. Most such cases can be easily diagnosed by abnormal patterns in spot shape, e.g. every second reflection has a non-Bragg streak associated with it. Apparent dense packing, 18% of the solvent, is likely to arise from random packing of molecules in alternative positions within the unit cell, where every second position is occupied. This randomness can be cross-correlated between cells, and this will produce a diffuse scattering. An alternative explanation is that you crystallised a proteolitic fragment of your protein. Zbyszek Otwinowski Dear all i have a small query to ask and seek your suggestions: I have collected a data for a protein with 324 residues and processed at its best in P212121. So Matthews suggest 1 mol in ASU with expected Mol. weight of 43 kDa with sovent content of 58% and 2 mol./ASU with 18% solvent content. However the data suggest possibility of translational NCS so i think i should ask for two molecules so that both get corrected for NCS. However for 2 mol./ASU, Matthewssuggests a total mol. weight of 52 kDa. So how to decide which way to proceed for MR? Thanks Monica Zbyszek Otwinowski UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd. Dallas, TX 75390-8816 Tel. 214-645-6385 Fax. 214-645-6353
[ccp4bb] Industry Postdoc Position - Boston
H3 Biomedicine seeks to recruit an outstanding postdoctoral candidate in structural biology. Our major area of research is RNA splicing in cancer. We have developed tool molecules targeting aberrant splicing in SF3B1 mutant cancers. Our goal is to understand the structural basis for aberrant splicing in this mutant background and use the tool molecules as a platform for structure based drug design. Relevant Review: http://www.nature.com/nrd/journal/v11/n11/full/nrd3823.html Qualifications The applicant must have a Ph.D. with a strong background in recombinant protein expression, crystallization, and structure determination. Experience in biophysics is also desirable. Candidate should be highly motivated and results driven. Strong organizational and interpersonal skills are desired. Funding is for three years with competitive salary and benefits. The laboratory is newly equipped. The candidate will be integrated into a dynamic and collaborative work environment with: * Regular interactions with mentor and potential for external academic advisor as appropriate * Regular presentations at Scientific Founders Meeting to the H3 Biomedicine senior scientists and our academic founders Stuart Schreiber, PhD and Todd Golub, MD. * Presentation/publication of research at major scientific conferences/journals * Potential for transition into full-time employment should appropriate positions be available at the end of the postdoctoral position To apply for this position, submit your resume at: http://www.h3biomedicine.com/career/postdoctoral-fellows-oncology-research Or e-mail: nicholas_lar...@h3biomedicine.com -- [This e-mail message may contain privileged, confidential and/or proprietary information of H3 Biomedicine. If you believe that it has been sent to you in error, please contact the sender immediately and delete the message including any attachments, without copying, using, or distributing any of the information contained therein. This e-mail message should not be interpreted to include a digital or electronic signature that can be used to authenticate an agreement, contract or other legal document, nor to reflect an intention to be bound to any legally-binding agreement or contract.]
Re: [ccp4bb] Kabat, insertion codes refinement
Hi David - Your input files for Refmac (I’m not sure about Buster) should have LINKR records of the form: LINKRGLY L 95 THR L 95A gap This has worked fine for me in the past. The file I happened to excerpt here was refined with Refmac 5.6.0117 a few years back, but I doubt this has changed since then. I’d say to make sure you have all the appropriate LINKRs present (typically located between SSBOND and CRYST1 records) and try again. Cheers, Jared -- Jared Sampson Xiangpeng Kong Lab NYU Langone Medical Center http://kong.med.nyu.edu/ On Jun 16, 2014, at 6:07 AM, Hargreaves, David david.hargrea...@astrazeneca.commailto:david.hargrea...@astrazeneca.com wrote: Dear CCP4bb, I’m refining an antibody structure which requires Kabat residue numbering with insertion codes. My setup of Refmac5 and Buster both break peptide bonds between some (not all) of the residues with insertion codes. I was wondering whether there is a special way of handling these residues in refinement? Thanks, David David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.commailto:name.surn...@astrazeneca.com Please consider the environment before printing this e-mail AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies. This email message, including any attachments, is for the sole use of the intended recipient(s) and may contain information that is proprietary, confidential, and exempt from disclosure under applicable law. Any unauthorized review, use, disclosure, or distribution is prohibited. If you have received this email in error please notify the sender by return email and delete the original message. Please note, the recipient should check this email and any attachments for the presence of viruses. The organization accepts no liability for any damage caused by any virus transmitted by this email. =
[ccp4bb] Grade other ligand tools was Re: ccp4 ligand tools..
... On 13/06/14 00:19, Ethan A Merritt wrote: Earlier this year for the first time I got back a validation report from the PDB for a deposited structure that included wwPDB validation of a ligand. This is great stuff. I approve. I am happy. Unfortunately the validation check reported problems with my ligand. This is bad. I am unhappy. ... On Fri, 13 Jun 2014, Paul Emsley wrote: In the mean-time you can use the Coot's Mogul plug-in to update the restraint information from cprodrg. Or of course, Just Use Grade (as you imply :-). Ethan, As you and Paul mentioned the use of Grade, I thought it might be useful to point out that as well as being included with BUSTER (so free for Academics) the Grade Web Server: http://grade.globalphasing.org/ This allows you to produce Grade dictionaries (for non-confidential ligands) without having to install BUSTER or CSDS (for the Mogul program). Grade generates geometrical restraints for novel small molecules. It does so by querying the Cambridge Structural Database (CSD) as a main source of restraint information (using Mogul), and then invoking quantum chemical procedures to obtain whatever information the CSD is unable to supply. Grade CIF restraint dictionaries can be used in Coot and REFMAC. In addition Grade and the Grade Web Server can produce SHELX restraint files. Mogul provides additional very useful validation measures for dihedrals and rings that go beyond the bond and angle results included in the wwPDB Structure Validation Report. These can be obtained using Mogul itself or using CSDS Mercury but I tend to get a bit lost using these. So we have added Mogul analysis to our buster-report tool and use colored 2D diagrams to quickly show the information. For details see http://www.globalphasing.com/buster/wiki/index.cgi?BusterReport For an example where Mogul validation metrics help to diagnose a ligand that has been mis-fit see: http://grade.globalphasing.org/tut/erice_workshop/1pmq_tutorial/ For difficult ligands BUSTER provides the facility to use a weighted Quantum Chemical or Force Field Energy in place of convential restraint terms. A recent advance is support for the MMFF94 force field using a helper program from OpenEye. This is particularly attractive as it is the force field used by many computational chemists. For details see http://www.globalphasing.com/buster/wiki/index.cgi?AutobusterLigandQM If you have concerns about Grade or other dictionaries then using MMFF94 instead provides a really good 2nd opinion. Hope that ccp4bb will forgive this bit of promotion but Grade can be used with CCP4 (and SHELX). Regards, Oliver | Dr Oliver Smart | | Global Phasing Ltd., Cambridge UK | | http://www.globalphasing.com/people/osmart/ |
Re: [ccp4bb] Hosed-Up X-Ray Structures: A Big Problem
Hi Tim, just to spice your words up with some numbers You may also want to note that constrained hydrogen positions are a crude approximation and only work with X-ray data where hydrogen atoms have little impact on the data. This contribution can be as large as 1.5% difference in R-factor (with vs without H), as shown in Figure 2 (page 19; On the contribution of hydrogen atoms to X-ray scattering): http://phenix-online.org/newsletter/CCN_2012_01.pdf Our comparison between hydrogen restraints and constraints (http://dx.doi.org/10.1107/S1600576713027659) report the greater quality of restraints vs. constraints when it comes to neutron data, where hydrogen atoms do matter. I just re-refined (phenix.refine) all neutron structures available in PDB (for which I could extract diffraction data without manual labor; 55 in total) with two ways of handling H (D and H/D) atoms: a) refine H individually, and b) using riding model for H (rotatable H are adjusted to fit the map). In terms of Rfree and Rwork I don't see a huge difference. However, using riding model results in less overfitting: http://cci.lbl.gov/~afonine/tmp/r_stats.pdf This is not surprising given typical quality of neutron data: average (all neutron entries in PDB) completeness of neutron data sets is 76%, while average completeness of comparable X-ray data sets is 94% (page 21): http://phenix-online.org/presentations/latest/2012_afonine_ecm27-final.pdf All the best, Pavel
[ccp4bb] Lysozyme crystals
Dear all Would anyone have nice images of lysozyme crystals in different space groups (monoclinic, triclinic and tetragonal)? Google wasn't particularly helpful... Thanks. Mohamed
[ccp4bb] Two Crystallography Job at the University of Michigan, Ann Arbor
The Stuckey Lab in the Life Sciences Institute at the University of Michigan, Ann Arbor campus has two jobs available. Please redistribute these postings however you see fit. Visit umjobs.org for additional information and to apply. Research Lab Specialist Intermediate\Associate http://umjobs.org/job_detail/97239/research_lab_specialist_intermediateassociate Job Opening # 97239 The Center for Structural Biology, within the Life Sciences Institute at the University of Michigan, seeks an experienced crystallographer to advance the research objectives of principal investigators in a wide variety of basic science fields and drug development. The Center includes: - A high-throughput cloning and expression laboratory for protein engineering - Protein expression and purification facilities for small- and large-scale protein production - Macromolecular crystallization and crystallography laboratories for solving crystal structures of biological molecules - X-ray facilities with membership access to high energy synchotron radiation (LS-CAT beam-lines at the Argonne National Laboratory) Staff scientists in the center will work under the direction of Jeanne A. Stuckey, Ph.D, Managing Director of CSB and Research Associate Professor, and work in collaboration with members of her lab and researchers who use the facilities. The successful candidate will apply their energy and expertise to cutting edge science and drug discovery by: - Designing and executing experiments in collaboration with others. - Working in an independent manner. - Aid in coordinating and managing projects on schedule - Maintain clear laboratory notebooks, perform quantitative analyses, and communicate findings through reports and summaries. - Provide detailed documentation so experiments can be replicated. - Follow laboratory best practices for safety and quality. - Being an excellent proactive communicator, both verbally in writing. - Carrying out innovative research resulting in co- and lead-authored publications on structures of therapeutic and biological importance. - Facilitating the utilization of protein structures by providing expert interpretation in order to enhance the design efforts of the medicinal and computational chemists. - Executing your grasp of modern cloning, expression and purification methodologies. - Developing novel crystallization techniques. - Performing crystallization screens. - Solving structures. RESEARCH FELLOW http://umjobs.org/job_detail/97198/research_fellowJob Opening # 97198 The Center for Structural Biology, within the Life Sciences Institute at the University of Michigan, seeks a post-doctoral candidate with crystallography experience, who is interested in developing cutting edge purification and crystallization technologies for RNA and RNA-Protein complexes. The Center for Structural Biology acts as the structural biology core for the Center for HIV RNA Studies (CRNA), which is a large consortium of researchers across the nation focusing on the structures, dynamics, interactions, and mechanisms of action of the HIV-1 RNA during the late phase of viral replication. Funding for these studies is provided by the National Institutes of General Medical Sciences, National Institutes of Health. The Center for Structural Biology includes: - A high-throughput cloning and expression laboratory for protein engineering - Protein expression and purification facilities for small- and large-scale protein production - Macromolecular crystallization and crystallography laboratories for solving crystal structures of biological molecules - X-ray facilities with membership access to high energy synchotron radiation (LS-CAT beam-lines at the Argonne National Laboratory) The Post-doctoral fellow will work under the direction of the Center Director and Managing Director, Drs. Janet Smith and Jeanne Stuckey, and work in collaboration with members of the CRNA. The successful candidate will apply their energy and expertise to cutting edge science by: - Designing and executing experiments in collaboration with others. Working in an independent manner. - Maintain clear laboratory notebooks, perform quantitative analyses, and communicate findings through reports and summaries. - Provide detailed documentation so experiments can be replicated. - Follow laboratory best practices for safety and quality. - Being an excellent proactive communicator, both verbally in writing. - Carrying out innovative research resulting in co- and lead-authored publications on structures of biological importance. - Executing your grasp of modern cloning, expression and purification methodologies. - Developing novel crystallization techniques. - Performing crystallization screens. - Solving and Refining structures. -- Rebecca Epstein Administrative Assistant Senior University of
Re: [ccp4bb] Kabat, insertion codes refinement
Insertion codes are a commonly used part of the PDB specification. It's odd that they wouldn't be supported correctly. To take another similar case, what would you say of a program that couldn't handle negative residue numbers as is commonly done with N-terminal purification tags? All sequences must start with 1? (Not all antibodies are isolated from natural sources. Some are from human-designed libraries for example, so they are every bit as engineered as something with as His tag stuck on the end.) Cheers, Eric On Jun 16, 2014, at 7:23 AM, Ed Pozharski wrote: There is no actual requirement to use Kabat numbering, you can avoid it alrogether. Some argue that L27A is actually 28th amino acid in the protein sequence, and labeling it as L27A is simply incorrect. I would suggest doing refinement with plain numbering (no insertion codes) and changing it only for the final model if needed for comparative analysis. Ed Sent on a Sprint Samsung Galaxy S® III Original message From: Hargreaves, David Date:06/16/2014 6:07 AM (GMT-05:00) To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Kabat, insertion codes refinement Dear CCP4bb, I’m refining an antibody structure which requires Kabat residue numbering with insertion codes. My setup of Refmac5 and Buster both break peptide bonds between some (not all) of the residues with insertion codes. I was wondering whether there is a special way of handling these residues in refinement? Thanks, David David Hargreaves Associate Principal Scientist _ AstraZeneca Discovery Sciences, Structure Biophysics Mereside, 50F49, Alderley Park, Cheshire, SK10 4TF Tel +44 (0)01625 518521 Fax +44 (0) 1625 232693 David.Hargreaves @astrazeneca.com Please consider the environment before printing this e-mail AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies. -- Eric Bennett, er...@pobox.com Always try to associate yourself with and learn as much as you can from those who know more than you do, who do better than you, who see more clearly than you. - Dwight Eisenhower
Re: [ccp4bb] Hosed-Up X-Ray Structures: A Big Problem
Hi, Tim, When we were working on our paper in 2011, refmac had a bug that always indicated in depostions that riding hydrogens were used, whether they were or not. Published methods did not always clarify this issue. Since we could not be definitive, we refrained from saying too much about it. However, then current refmac structures did have plenty of close non-bonded contacts as all-atom models, even though some (most? all?) of them were refined with riding hydrogens 'on'. We should look again with a recent sample of structures and find out. Your point is well taken that constrained hydrogen coordinates may not agree well where accurate data is available for hydrogen positions. One builds the best model that one can with the available data. PrimeX is intended for use with moderate resolution X-ray structures. Hydrogen positions are determined by the force field while heavier atom positions are refined to agree with the diffraction data and force field. Cheers, Jeff On Monday, June 16, 2014 7:14 AM, Tim Gruene t...@shelx.uni-ac.gwdg.de wrote: Dear Jeff, I would assume that clashing hydrogen atoms beome less and less an issue with current refinement programs, since those I am familiar with (refmac5 and phenix) both genereate constrained hydrogen atoms by default now, and it has been like this for quite some time - so the situation should become better for modellers. You may also want to note that constrained hydrogen positions are a crude approximation and only work with X-ray data where hydrogen atoms have little impact on the data. Our comparison between hydrogen restraints and constraints (http://dx.doi.org/10.1107/S1600576713027659) report the greater quality of restraints vs. constraints when it comes to neutron data, where hydrogen atoms do matter. Hydrogen positions are much more flexible than the usual riding atom model may imply. This may affect in silico simulations. Best, Tim On 06/16/2014 04:34 AM, Jeffrey Bell wrote: Hi, all, I am glad to see these matters being discussed. I think we all believe that protein crystallographers should be concerned with producing models that modelers and chemists can respect and use. Schrödinger spends a lot of time thinking about ligands; its refinement program, PrimeX, has a very simple way of handling ligand issues. All that a crystallographer has to do is get the charge and bond order right, and the force field then automatically does atom typing and generates all restraints. However, use of our cif library files brings up another matter that must be understood first. Use of PrimeX, even at low resolution, involves refinement of all hydrogen atom positions. The Richardsons have abundantly demonstrated how important hydrogen coordinates are to accurate model building. This matter of hydrogen coordinate refinement is closely connected to the editorial that started this thread. Computational chemistry in drug discovery, and elsewhere, uses all-atom models. When you add hydrogen atoms to most models in the PDB, many chemically-impossible overlaps of atoms result (see Acta Cryst. 2012, D68, 935-952 for more information; ask me for a copy). This issue is almost as much of an annoyance for computational chemists as bad ligand geometry because they see it in almost every structure. If anyone would like to try PrimeX, either for ligand restraint generation or refinement, please let me know. Schrödinger offers academic institutions one year of free access, which may be renewed on a case-by-case basis. Crystallographers at companies will also qualify for a free evaluation trial. Cheers, Jeff Bell PrimeX developer Schrödinger, Inc. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A
