Re: [ccp4bb] Ligand geometry
Hi Uma, How different are your NADs optimised in Refmac and Coot? Are you sure you are using the same geometric restraints? Coot has to know where Refmac's restraint files are. This info is passed through an environment setting on your computer (I don't know the name by hart. Anyone?). Are you using Windows, Linux or OSX or something else? You can try to find more details about geometric outliers by checking Refmac's log file. That way you may find which specific bond/angle is the problem. Cheers, Robbie Date: Sat, 28 Apr 2012 11:47:58 -0400 From: rosiso2...@gmail.com Subject: [ccp4bb] Ligand geometry To: CCP4BB@JISCMAIL.AC.UK Dear All: I use Refmac5 to refine my model. After the run, I check the model quality by Coot. Here is the problem: In Coot, the ligand - NAD, has bad geometry as indicated by a big red bar. While the geometry of NAD fit nicely with the electron density. If I use refine tools (i.e. regularize Zone or real space refine zone), the geometry of NAD turns to perfec with bond, angle and so on. But the ligand slightly turn away from the electron density map. If I run Refmac5 again with this modified model, the NAD turns back, fit nice to electron density, but gives red bar in coot geometry. The Refinment Parameters in Refmac5 is set @ use automatic weight and use experinmental sigmals to weight X-ray terms. Thank you for advice and comments Ros
Re: [ccp4bb] Ligand geometry
Hmm, what are the perfect bonds, angles for NAD in your protein? remember that reactive groups can be in a stressed conformation, compared to ideal in vacuo conformations. As part of their functon. anyway, you'll have to check the restraints definition file (.cif). Bond lengths and angles are usually ok, but make sure only chiral atoms are defined as chiral, others need to be deleted or defined as both. Check that the torsion angles make chemical sense, especially the repetition factor for rotatable bonds. Rotatable bonds next to aromatic rings are often problematic. You might need to set high sigmas, and repetition factors (x/360 degrees). On the other hand, you say that refmac behaves well, so the weighting scheme can't be far off. Cheers, Hans. And Uma Ratu schreef: Dear All: I use Refmac5 to refine my model. After the run, I check the model quality by Coot. Here is the problem: In Coot, the ligand - NAD, has bad geometry as indicated by a big red bar. While the geometry of NAD fit nicely with the electron density. If I use refine tools (i.e. regularize Zone or real space refine zone), the geometry of NAD turns to perfec with bond, angle and so on. But the ligand slightly turn away from the electron density map. If I run Refmac5 again with this modified model, the NAD turns back, fit nice to electron density, but gives red bar in coot geometry. The Refinment Parameters in Refmac5 is set @ use automatic weight and use experinmental sigmals to weight X-ray terms. Thank you for advice and comments Ros
Re: [ccp4bb] Ligand geometry
I think you will find the dictionaries for coot and refmac are different.. REFMAC default dictionary will $CLIBD/monomers/n/NAD.cif Good knows where coot finds its dictionaries - Paul? 1) check the REFMAC restraints in that dictionary are sensible - spec, are the planes and chiral centres as you would expect 2) read the REFMAC dictionary into coot and see what happens.. do you still have the problem. 3) check the REFMAC log file for what it thinks are outliers after the COOT refinement - I reset the geometric monitoring parameters - see on the GUI - to list things with errors of 5 Sigma say ( I think the default is 10 Sigma) 4) If there are difference complain.. Eleanor On 29 April 2012 10:14, H. Raaijmakers hraaijmak...@xs4all.nl wrote: Hmm, what are the perfect bonds, angles for NAD in your protein? remember that reactive groups can be in a stressed conformation, compared to ideal in vacuo conformations. As part of their functon. anyway, you'll have to check the restraints definition file (.cif). Bond lengths and angles are usually ok, but make sure only chiral atoms are defined as chiral, others need to be deleted or defined as both. Check that the torsion angles make chemical sense, especially the repetition factor for rotatable bonds. Rotatable bonds next to aromatic rings are often problematic. You might need to set high sigmas, and repetition factors (x/360 degrees). On the other hand, you say that refmac behaves well, so the weighting scheme can't be far off. Cheers, Hans. And Uma Ratu schreef: Dear All: I use Refmac5 to refine my model. After the run, I check the model quality by Coot. Here is the problem: In Coot, the ligand - NAD, has bad geometry as indicated by a big red bar. While the geometry of NAD fit nicely with the electron density. If I use refine tools (i.e. regularize Zone or real space refine zone), the geometry of NAD turns to perfec with bond, angle and so on. But the ligand slightly turn away from the electron density map. If I run Refmac5 again with this modified model, the NAD turns back, fit nice to electron density, but gives red bar in coot geometry. The Refinment Parameters in Refmac5 is set @ use automatic weight and use experinmental sigmals to weight X-ray terms. Thank you for advice and comments Ros
Re: [ccp4bb] Ligand geometry
Quasi on-topic rant: I would advice against using the 'both' option for any well defined ligand. It's a hack to avoid thinking about which atom belongs where and it allows you to be inconsistent. This makes it difficult for others to use your model, because aligning atoms of ligands becomes needlesly complicated. To the eye an oxygen is an oxigen, to a computer O1 is different from O2. Just stick to the definition given by the PDB (see Ligand Expo). It's there for a reason. Cheers, Robbie Date: Sun, 29 Apr 2012 11:14:01 +0200 From: hraaijmak...@xs4all.nl Subject: Re: [ccp4bb] Ligand geometry To: CCP4BB@JISCMAIL.AC.UK Hmm, what are the perfect bonds, angles for NAD in your protein? remember that reactive groups can be in a stressed conformation, compared to ideal in vacuo conformations. As part of their functon. anyway, you'll have to check the restraints definition file (.cif). Bond lengths and angles are usually ok, but make sure only chiral atoms are defined as chiral, others need to be deleted or defined as both. Check that the torsion angles make chemical sense, especially the repetition factor for rotatable bonds. Rotatable bonds next to aromatic rings are often problematic. You might need to set high sigmas, and repetition factors (x/360 degrees). On the other hand, you say that refmac behaves well, so the weighting scheme can't be far off. Cheers, Hans. And Uma Ratu schreef: Dear All: I use Refmac5 to refine my model. After the run, I check the model quality by Coot. Here is the problem: In Coot, the ligand - NAD, has bad geometry as indicated by a big red bar. While the geometry of NAD fit nicely with the electron density. If I use refine tools (i.e. regularize Zone or real space refine zone), the geometry of NAD turns to perfec with bond, angle and so on. But the ligand slightly turn away from the electron density map. If I run Refmac5 again with this modified model, the NAD turns back, fit nice to electron density, but gives red bar in coot geometry. The Refinment Parameters in Refmac5 is set @ use automatic weight and use experinmental sigmals to weight X-ray terms. Thank you for advice and comments Ros
Re: [ccp4bb] Anisotropic diffraction
If the data set had P6 symmetry before anisotropic scaling it would keep that symmetry afterwards. If it was only P2 symmetry before, it certainly would not have P6 afterwards. Any anisotropic scaling I've seen constrains the anisotropy to the lattice symmetry so symmetry cannot be degraded via its application. If your data set had, in principle, P6 symmetry but was expressed in a lower symmetry asymmetric unit and contained nonsymmetry-conforming noise before anisotropic scaling it would also contain broken symmetry afterwards. The higher symmetry was not lost, it was never there to begin with. Dale Tronrud On 4/28/2012 12:06 AM, Zhijie Li wrote: Hi, My first thought was same with David: the truncation won't change the crystal's space group. The symmetry of the crystal is reflected by the symmetry of the amplitudes of many many reflections across all resolutions. Ellipsoidal truncation itself only removes some very weak reflections from the outer shells. The remaining reflections will still have a good number of reflections carrying the symmetry of the crystal. However a second thought on the anisotropic scaling and B-factor correction led me to this scenario: suppose we have a crystal that's really P6, but we have cowardly indexed it to a lower space group P2, with the 2-fold axis, b, coinciding the real 6-fold axis. By losing the a=c restrain, the anisotropic scaling along H and L now may not be strictly equal (for example, could be caused by outliers that would have been identified and filtered out if indexed correctly as P6), resulting in the loss of the 6-fold symmetry in the scaled dataset. Apparently this is an artifact due to an improper SG assignment before the anisotropic scaling and B-factor correction. Just some crazy thoughts. Please correct me if I am wrong. BTW, to Theresa: an very informative introduction on ellipsoidal truncation and anisotropic scaling can be found here: http://services.mbi.ucla.edu/anisoscale/ -- From: Theresa Hsu theresah...@live.com Sent: Friday, April 27, 2012 3:18 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Anisotropic diffraction Dear crystallographers A very basic question, for anisotropic diffraction, does data truncation with ellipsoidal method change the symmetry? For example, if untruncated data is space group P6, will truncated data index as P622 or P2? Thank you. Theresa
Re: [ccp4bb] Anisotropic diffraction
Hi Chen, I see your point now. Yes, I agree that the 80:20 method (or 75:25 as stated in the paper, http://www.mail-archive.com/ccp4bb@dl.ac.uk/msg01063.html) is a very useful technique. The fact that it does not take much effort or lead to uncertainties makes it very worth trying. Here I add my two cents: when performing such optimizations, try both seeding and no-seeding if quantity of protein permits. If protein quantity is limited and the crystals are reluctant to appear under the original condition, then seeding is always a good idea. Zhijie -- From: chen c chenc...@gmail.com Sent: Sunday, April 29, 2012 6:09 PM To: Zhijie Li zhijie...@utoronto.ca Subject: Re: [ccp4bb] Anisotropic diffraction I accept your advice. In fact, this is the first time I am involved in anisotropic issue. And I learned a lot from all the above discussion. However, the 80:20 optimization method(an example of long-tail theory) rather than surface mutation is what I want to emphasis in my last email. As illustrated in the attached pdf, it defenitely deserve a try. One more thing to mention is that this very 80:20 method can be very versatile and useful in optimising macromolecular crystals in case of issues more than anistropic problem. best regards chen 2012/4/30 Zhijie Li zhijie...@utoronto.ca: Hi Chen, It is a reality that a usable protein dataset could take years of hard work to obtain. Compared to problems such as twining, bad diffraction patterns, excessive mosaicity, low resolution, weak, noisy anomalous signal, etc., anisotropic diffraction should probably be regarded most benign. There is nothing wrong with publishing a properly treated anisotropic dataset. Then why risking another year trying to find a better mutant? There is no guarantee that the mutants would work better, or even work. Unless the crystal is naturally isotropic, such as that it is in a cubic space group, the diffraction of protein crystals will probably always be more or less anisotropic. This only reflects the fact that the crystal packing is indeed anisotropic, consequently the movements of the Unit cell is anisotropic. It does not mean that there would be defects in the resulting structure. For instance, a 3A isotropic dataset, and a 2.5-2A anisotropic dataset, which one is going to give a better description of the protein? Zhijie -- From: chen c chenc...@gmail.com Sent: Sunday, April 29, 2012 5:03 AM To: Zhijie Li zhijie...@utoronto.ca Subject: Re: [ccp4bb] Anisotropic diffraction If we might be able to eliminate the anisotropic diffraction issue, which might mean and reflect defect in structure. Why not just restrain our thought to solve this question by data processing, etc? chen 2012/4/28 Zhijie Li zhijie...@utoronto.ca: Hi Cheng, This paper looks quite irrelevant to Theresa's question. Zhijie -- From: chen c chenc...@gmail.com Sent: Friday, April 27, 2012 10:28 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Anisotropic diffraction Birtley and Curry used a novel optimization method, in their paper Crystallization of foot-and-mouth disease virus 3C protease: surface mutagenesis and a novel crystal-optimization strategy, which might be inspiring for you. 在 2012年4月28日 上午3:21，David Schuller dj...@cornell.edu 写道： Anisotropic truncation should have no effect on the space group symmetry. On 04/27/12 15:18, Theresa Hsu wrote: Dear crystallographers A very basic question, for anisotropic diffraction, does data truncation with ellipsoidal method change the symmetry? For example, if untruncated data is space group P6, will truncated data index as P622 or P2? Thank you. Theresa -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu -- Cheng Chen, Ph.D. Candidate Laboratory of Structural Biology Life Science Building,Tsinghua University Beijing 100084 China Tel:+86-10-62772291 Fax:+86-10-62773145 E-mail:che...@xtal.tsinghua.edu.cn 北京市海淀区清华大学生命科学馆201-212室 邮编：100084 -- Cheng Chen, Ph.D. Candidate Laboratory of Structural Biology Life Science Building,Tsinghua University Beijing 100084 China Tel:+86-10-62772291 Fax:+86-10-62773145 E-mail:che...@xtal.tsinghua.edu.cn 北京市海淀区清华大学生命科学馆201-212室 邮编：100084 -- Cheng Chen, Ph.D. Candidate Laboratory of Structural Biology Life Science Building,Tsinghua University Beijing 100084 China Tel:+86-10-62772291 Fax:+86-10-62773145 E-mail:che...@xtal.tsinghua.edu.cn 北京市海淀区清华大学生命科学馆201-212室 邮编：100084
