Re: [ccp4bb] Fwd: [ccp4bb] adjusting bad Ramachandran angles
Jeremy, apparently I was unclear (and possibly moving off-topic) since I got other emails off list about this. I did not mean to imply that I would use Ramachandran restraints to fix a register error since this obviously needs to be addressed in real space. Rather, inspection of the Ramachandran plot can help to highlight serious errors in your structure - or, of course, in other people's structures. I would say that an outlier residue is something that should be noticed, and then a decision must be made about whether is needs to be fixed. The Ramachandran plot has a physical basis, and there is an energetic cost to violating it in the same way that there is a cost for poor side chain rotamers, non-ideal hydrogen bond geometry, etc. If I encounter a Ramachandran outlier (or a poor rotamer, odd-looking hydrogen bond, or any other unusual feature of my model), I examine it to see if it is supported by the electron density and the chemical environment or if a lower-energy conformation is more appropriate. If the electron density is ambiguous, I will choose an allowed conformation but will not restrain it. I see this as equivalent to flipping a His side chain to form proper hydrogen bonds - although we can't distinguish between the carbon and nitrogen atoms in the ring based on electron density, chemical knowledge tells us the one conformation is right and one is wrong. But certainly many (if not most) of the structures that I have solved or signed off on have had a small number of Ramachandran outliers. kmj On Thu, Feb 26, 2015 at 6:42 PM, Jeremy Tame jt...@tsurumi.yokohama-cu.ac.jp wrote: Dear Kevin Please re-read my earlier email paying particular attention to the words explicitly and expressly. To my mind, the notion of moving a Ramachandran outlier by moving a dot across a graph (which you feel is a danger) is identical to expressly forcing that residue into a favoured Ramachandran region. What you suggest doing is pretty much what I do do. I pay a great deal of attention to the Ramachandran plot at every stage of refinement, noting outliers and looking for the sorts of problems you mention, but I do not attempt to correct register errors (for example) by restraining phi/psi angles! Gert and Robbie have explained clearly why phi/psi angles do not lend themselves easily to fitting, and it seems best (to me at least) to leave the Ramachandran plot as an unfudged indicator of the interesting/problematical residues, not only for subsequent PDB users but for the crystallographer him/herself during refinement. I think we are in perfect agreement up to this point. Where we differ is in your apparent view that an outlier residue must be fixed. The first structure I ever solved had one residue with unusual backbone geometry, and I was given substantial grief from a modeller in the group over it - but it turned out to be a crucial functional residue at a hinge which flips between open and closed forms. Of course, it can be a pain explaining to a reviewer/editor/supervisor/PDB-bod why your model is flagged by validation software, but sometimes this is where the interesting bits are. If we have such a strongly defined preconception of what proteins look like then isn't the experimental data becoming undervalued? There are many parts of proteins that are not modelled well by single conformations, and the expectation that every atom will sit nicely in one place and behave is sometimes a little unrealistic. Dear Judit I am sure there are cases at AstraZeneca where repeated refinement of a well-known protein with umpteen different test drugs is expedited by the tools you suggest, but the aim of such experiments is rather different from an initial structure determination. I suspect that if Michael Murphy had wanted to know how to use a prior high-resolution model as a set of restraints for later lower resolution refinements then he would have framed his original question rather differently. When you say many crystallographers now prefer better quality structures over pedantic validation tools... I have to wonder what is meant by quality, but that is perhaps opening too large a can of worms. Basically though, beauty and truth are not synonymous, whether or not we regard protein crystallography as a video game where the person with the lowest R-factor wins. best wishes to all! Jeremy the Pedant Begin forwarded message: *From: *Kevin Jude kj...@stanford.edu *Date: *February 27, 2015 3:25:24 AM GMT+09:00 *To: *Jeremy Tame jt...@tsurumi.yokohama-cu.ac.jp *Cc: *CCP4BB@jiscmail.ac.uk *Subject: **Re: [ccp4bb] adjusting bad Ramachandran angles* I think the Ramachandranplot should be used in the refinement and rebuilding process - a Ramachandran outlier is a flag that that region of the model needs a closer look, and the fix may be more complicated than simply rotating a peptide. Maybe a C-beta is pointing the wrong way, maybe there is
[ccp4bb] different Kd and Km value
Hi I have a protein with two substrate. when I am doing the binding studies with the two substrate separately, I am finding one of the substrate to have similar kd and Km. however the km and kd values are almost 30 fold different for the other substrate. it binds 30 fold more tightly then you can think based on Km. I don't know how to explain this. Km and Kd can be different but does any one have seen that much difference in Kd and Km? Thank you Dhiraj
Re: [ccp4bb] RMSD of dimers
Doesnt pisa give you some of this information? It lists all likely homodimers and I think gives RMSD too Eleanor On 24 February 2015 at 22:00, Thomas Holder thomas.hol...@schrodinger.com wrote: Hi Dan, gaps/insertions should be no problem for PyMOL's rms_cur command, as long as chain identifiers match (and all other atomic identifiers!). See http://pymolwiki.org/index.php/Fit for a description of the matchmaker argument. Cheers, Thomas On 24 Feb 2015, at 16:30, D Bonsor dbon...@ihv.umaryland.edu wrote: I have a family of homodimers (denoted A1B1, A2B2, A3B3...) which I have superimposed using Chain A. Several programs will produce the RMSD of Chain A2, A3, A4... to Chain A1. However, I would like to know the RMSDs of Chain B2, B3, B4... to Chain B1 when I have superimposed the structures relative to Chain A. I have tried using Pymol though there are gaps/insertions so rms/rms_cur will not work. Does anyone else have any other suggestions? Thanks in advance, Dan -- Thomas Holder PyMOL Principal Developer Schrödinger, Inc.
[ccp4bb] Postdoc position in structural biology of a novel bifunctional bacterial transporter at Diamond/RCaH -closing date for applications 15th March
Dear All We have a vacancy for an enthusiastic postdoctoral researcher to join our group within the Research Complex at Harwell to investigate the structure and function of a novel bacterial integral membrane complex involved in innate immune evasion using primarily single particle electron cryo microscopy in combination with X-ray crystallography. Full details and how to apply can be found here: http://www.diamond.ac.uk/Careers/Vacancies/All/DIA1004-CH.html IF you have informal enquires don’t hesitate to contact me by email Martin -- -- This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
[ccp4bb] CCP4 6.5 Windows-32
Dear CCP4 Users, CCP4 Core Group would like to estimate the demand for 32-bit Windows distribution of CCP4 Release 6.5. Therefore, could you please help it by replying to this e-mail without changing the subject _/*if and only if*/__/*you have to use CCP4 on a 32-bit Windows PC*/__/. /_ Please do not put any message in as only the total number of replies will be counted, and please do not reply to all so that your e-mail does not increase traffic on CCP4 BB. */HOW TO IDENTIFY WHETHER YOUR WINDOWS IS 32 OR 64-BIT/* - open Control Panel - go to System and Security - click System - read System type. It should say something like XX-bit Operating System, YYY processor /*WHY WE ASK YOU TO GIVE YOUR FEEDBACK*/ Although it does not scale linearly with the number of supported OSes, each OS requires separate manual effort to make a release and support it through updates. Two years ago we ran a similar query regarding 32-bit Mac OSX, and only a handful of users reported the use of 32-bit Mac hardware. We took the decision not to provide 32-bit Mac OSX binaries then because that was clearly uneconomical. At that point we were already 5 years behind Apple's own decision not to produce and support 32-bit hardware and OSes any longer. Today, we observe a similar situation for Linux and MS Windows. According to CCP4 download stats, only 10% of CCP4 Linux users have 32-bit machines. Up to this point, we did not release 32-bit 6.5 package for MS Windows, and only a few users have asked for it. In difference of Mac OSX, 32-bit versions of MS Windows and Linuxes are still provided by the respective vendors. Therefore, we will not drop 32-bit CCP4 packages for these systems if actual demand proves to be significant. /*PLEASE NOTE:*//* */ 1. Using 32-bit CCP4 packages becomes increasingly disadvantageous, because some programs may require 64-bit memory space, depending on data and structure size. 2. It is highly unlikely that a 5-year old and newer computer hardware is not a 64-bit one. The installed OS may be, however, both 32 and 64-bit. Please see the previous note if you are choosing between the systems. Many thanks for your help, Eugene Krissinel.
Re: [ccp4bb] RMSD of dimers
No, the question seems to be slightly different. While one can use either CCP4's SSM or GESAMT to superpose 2 dimers, these algorithms would balance RMSD between both chains and produce something optimal for both chains. What is required, however, is optimal superposition on one pair of chains (one from each dimer) but measuring RMSD on the other pair of chains from same dimers. Nothing impossible in principle but no corresponding option in general-purpose aligners (this requires something like masks/selections for residues to superimpose and residues to measure the RMSD on). If this were a common and frequent problem, we could have it implemented in SSM/GESAMT. Eugene On 26/02/2015 12:47, Eleanor Dodson wrote: Doesnt pisa give you some of this information? It lists all likely homodimers and I think gives RMSD too Eleanor On 24 February 2015 at 22:00, Thomas Holder thomas.hol...@schrodinger.com mailto:thomas.hol...@schrodinger.com wrote: Hi Dan, gaps/insertions should be no problem for PyMOL's rms_cur command, as long as chain identifiers match (and all other atomic identifiers!). See http://pymolwiki.org/index.php/Fit for a description of the matchmaker argument. Cheers, Thomas On 24 Feb 2015, at 16:30, D Bonsor dbon...@ihv.umaryland.edu mailto:dbon...@ihv.umaryland.edu wrote: I have a family of homodimers (denoted A1B1, A2B2, A3B3...) which I have superimposed using Chain A. Several programs will produce the RMSD of Chain A2, A3, A4... to Chain A1. However, I would like to know the RMSDs of Chain B2, B3, B4... to Chain B1 when I have superimposed the structures relative to Chain A. I have tried using Pymol though there are gaps/insertions so rms/rms_cur will not work. Does anyone else have any other suggestions? Thanks in advance, Dan -- Thomas Holder PyMOL Principal Developer Schrödinger, Inc.
Re: [ccp4bb] RMSD of dimers
Oops, sorry: lsqman gives you the rmsd between pairs not moleman. Additional comment: PDBSET should also give you the rmsd between B1 abd B2 using the coordinates generated by lsqman. Phil Philippe BENAS, Ph.D. X-ray diffraction and computing facilities manager Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18 De : Philippe BENAS philippe_be...@yahoo.fr À : CCP4BB@JISCMAIL.AC.UK Envoyé le : Jeudi 26 février 2015 15h00 Objet : Re: [ccp4bb] RMSD of dimers Dear CCP4bbers, If I remember correctly you can do this in LSQMAN (EXPLICIT superimposition command if I remember well): the superimposition is made between residues of chain A2 onto those of chain A1, then you apply the rotation/translation to all the A2B2 dimer and moleman gives you the rmsd between the two pairs of dimers. Alternatively, I think LSQMAN allows you to calculate the rmsd between B1 and B2 after having applied the transformation to the all dimer.Have a look on GJK-DVD pages at http://xray.bmc.uu.se/usf/ . And finally isn't Coot doing this as well (using command lines) ? I think I did this once... HTS,Philippe Philippe BENAS, Ph.D. X-ray diffraction and computing facilities manager Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18 De : Eugene Krissinel eugene.krissi...@stfc.ac.uk À : CCP4BB@JISCMAIL.AC.UK Envoyé le : Jeudi 26 février 2015 14h17 Objet : Re: [ccp4bb] RMSD of dimers No, the question seems to be slightly different. While one can use either CCP4's SSM or GESAMT to superpose 2 dimers, these algorithms would balance RMSD between both chains and produce something optimal for both chains. What is required, however, is optimal superposition on one pair of chains (one from each dimer) but measuring RMSD on the other pair of chains from same dimers. Nothing impossible in principle but no corresponding option in general-purpose aligners (this requires something like masks/selections for residues to superimpose and residues to measure the RMSD on). If this were a common and frequent problem, we could have it implemented in SSM/GESAMT. Eugene On 26/02/2015 12:47, Eleanor Dodson wrote: Doesnt pisa give you some of this information? It lists all likely homodimers and I think gives RMSD too Eleanor On 24 February 2015 at 22:00, Thomas Holder thomas.hol...@schrodinger.com wrote: Hi Dan, gaps/insertions should be no problem for PyMOL's rms_cur command, as long as chain identifiers match (and all other atomic identifiers!). See http://pymolwiki.org/index.php/Fit for a description of the matchmaker argument. Cheers, Thomas On 24 Feb 2015, at 16:30, D Bonsor dbon...@ihv.umaryland.edu wrote: I have a family of homodimers (denoted A1B1, A2B2, A3B3...) which I have superimposed using Chain A. Several programs will produce the RMSD of Chain A2, A3, A4... to Chain A1. However, I would like to know the RMSDs of Chain B2, B3, B4... to Chain B1 when I have superimposed the structures relative to Chain A. I have tried using Pymol though there are gaps/insertions so rms/rms_cur will not work. Does anyone else have any other suggestions? Thanks in advance, Dan -- Thomas Holder PyMOL Principal Developer Schrödinger, Inc.
Re: [ccp4bb] RMSD of dimers
Dear CCP4bbers, If I remember correctly you can do this in LSQMAN (EXPLICIT superimposition command if I remember well): the superimposition is made between residues of chain A2 onto those of chain A1, then you apply the rotation/translation to all the A2B2 dimer and moleman gives you the rmsd between the two pairs of dimers. Alternatively, I think LSQMAN allows you to calculate the rmsd between B1 and B2 after having applied the transformation to the all dimer.Have a look on GJK-DVD pages at http://xray.bmc.uu.se/usf/ . And finally isn't Coot doing this as well (using command lines) ? I think I did this once... HTS,Philippe Philippe BENAS, Ph.D. X-ray diffraction and computing facilities manager Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr URLs: http://lcrbw.pharmacie.univ-paris5.fr/ , http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18 De : Eugene Krissinel eugene.krissi...@stfc.ac.uk À : CCP4BB@JISCMAIL.AC.UK Envoyé le : Jeudi 26 février 2015 14h17 Objet : Re: [ccp4bb] RMSD of dimers No, the question seems to be slightly different. While one can use either CCP4's SSM or GESAMT to superpose 2 dimers, these algorithms would balance RMSD between both chains and produce something optimal for both chains. What is required, however, is optimal superposition on one pair of chains (one from each dimer) but measuring RMSD on the other pair of chains from same dimers. Nothing impossible in principle but no corresponding option in general-purpose aligners (this requires something like masks/selections for residues to superimpose and residues to measure the RMSD on). If this were a common and frequent problem, we could have it implemented in SSM/GESAMT. Eugene On 26/02/2015 12:47, Eleanor Dodson wrote: Doesnt pisa give you some of this information? It lists all likely homodimers and I think gives RMSD too Eleanor On 24 February 2015 at 22:00, Thomas Holder thomas.hol...@schrodinger.com wrote: Hi Dan, gaps/insertions should be no problem for PyMOL's rms_cur command, as long as chain identifiers match (and all other atomic identifiers!). See http://pymolwiki.org/index.php/Fit for a description of the matchmaker argument. Cheers, Thomas On 24 Feb 2015, at 16:30, D Bonsor dbon...@ihv.umaryland.edu wrote: I have a family of homodimers (denoted A1B1, A2B2, A3B3...) which I have superimposed using Chain A. Several programs will produce the RMSD of Chain A2, A3, A4... to Chain A1. However, I would like to know the RMSDs of Chain B2, B3, B4... to Chain B1 when I have superimposed the structures relative to Chain A. I have tried using Pymol though there are gaps/insertions so rms/rms_cur will not work. Does anyone else have any other suggestions? Thanks in advance, Dan -- Thomas Holder PyMOL Principal Developer Schrödinger, Inc.
Re: [ccp4bb] Skin on drops
Hi Adam, The diffraction improved from 8 Angstrom to 2.6 Angstrom. But, I had also removed the His-tag from the protein, so I am not sure how much was the role of this step and how much was it because of the addition of DTT. Two variables in one experiment :). -Gyan On Wed, Feb 25, 2015 at 4:18 PM, Adam Brummett adam-brumm...@uiowa.edu wrote: Gyan, With the addition of DTT to remove the skin, did you see an increase in the resolution with the skin no longer present compared to with it still there? -Adam On Feb 25, 2015, at 11:15 AM, Gyanendra Kumar gyanendr...@gmail.com wrote: Adding DTT in your protein buffer or crystallization solution may also help. You could try increasing amounts of DTT/BME/TCEP in your crystallization solution and find a balance between reduction of skin formation vs getting crystals. Adding 2mM DTT in my protein buffer helped me get rid of much of the skin on the drop in which the crystals were tightly embedded. -Gyan On Wed, Feb 25, 2015 at 3:00 AM, Han Remaut han_c...@yahoo.co.uk wrote: Dear Ulrike, you could try avoid the drop-air interface by overlying sitting drops with silicone oil or a 50/50 silicon/paraffin oil mixture. Note that this will alter the kinetics with which your drops reach equilibrium, and hence may alter your ability to get crystals of the protein. Batch crystallization under oil is another option of course. Adding some alcohols (5-10% EtOH, isopropanol) or detergent (0.5 mM LDAO for example) in your crystallization conditions may also be something to consider. If none of these work, I'd concentrate on harvesting the crystals from the skin. I tend to cut these open from the side, flip over the skin so that one has better access to the crystals that generally are associated with the inner face of the skin. You can try peel the crystals off the skin, or cut out a piece of skin surrounding a crystal. That will not hurt diffraction quality of the crystals. Hope that helps. Han On 25 Feb 2015, at 09:34, Ulrike Demmer wrote: Dear crystallographers, I am trying to crystallize a soluble protein which tends to form aggregates. The crystallization condition is 20% PEG 3350 + 0,2 M Na-Formate. During the crstallization process a thick skin is formed on top of the sitting-drops. As well the crystals are buried in precipitate. Before I start harvesting I try to remove the skin but still it is hardly possible to get any crystals out of these drops. Any suggestions how to avoid the formation of skin on crystallization drops ? Cheers, Ulrike Han Remaut, PhD Laboratory of Structural Molecular Microbiology VIB / Vrije Universiteit Brussel Building E4, Pleinlaan 2 1050 Brussel han.rem...@vib-vub.be tel. +32-2-629 1923 / +32-499 708050 http://www.vib.be/en/research/scientists/Pages/Han-Remaut-Lab.aspx -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 --- -- Gyanendra Kumar, PhD St. Jude Children's Research Hospital, Department of Structural Biology, 262, Danny Thomas Place, MS-311 Memphis, TN 38105 Phone: 901-595-3839 Cell: 631-875-9189 ---
Re: [ccp4bb] adjusting bad Ramachandran angles
I think the Ramachandranplot should be used in the refinement and rebuilding process - a Ramachandran outlier is a flag that that region of the model needs a closer look, and the fix may be more complicated than simply rotating a peptide. Maybe a C-beta is pointing the wrong way, maybe there is a register error. The danger is that people will treat a Ramachandran outlier by moving a dot across a graph without addressing the underlying structural problem. kmj On Wed, Feb 25, 2015 at 8:37 PM, Jeremy Tame jt...@tsurumi.yokohama-cu.ac.jp wrote: I think Goodhart's Law applies here (see the Wikipedia page): When a measure becomes a target, it ceases to be a good measure. From memory I believe Randy Read and George Sheldrick have commented that Ramachandran plots are a good measure of structure quality, and therefore should not be used explicitly at the modelling stage. Some residues may be difficult if they have more than one backbone conformation or are just mobile, but expressly holding them in favoured regions of the Ramachandran plot is not a good idea. The most interesting proteins are of course enzymes, and the Ramachandran outliers are often among the most interesting active site residues. So you may be trying to eliminate something which is actually more important than a low Rfactor. The idea of limiting data use may seem counter-intuitive, but to take another example from economics, John Cowperthwaite was in charge of Hong Kong's financial affairs in the 1960s. He attributed the success of the economy under his tenure to his adamant refusal to collect any economic data whatsoever! On Feb 26, 2015, at 2:08 AM, Michael Murphy wrote: Does anyone know of a way to adjust Ramachandran angles so that they fall within the preferred range? Either in Coot or possibly some online server? I have been trying to do it manually without much success, I was wondering whether there might another way to do it. -Thanks
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Hello everyone A Symposium with a Spring School is being organized here in Hamburg: http://www.cssb-hamburg.de see Events Topic of the Symposium is Systems biology and Infection biology Topics in the Spring School are various Methods and Techniques, especially the EM / Single Particle. I am involved in crystallization of membrane proteins in cubic phase. A really good value for students is being offered: Symposium , Spring School including Food and Housing only for a nominal contribution of 150 € There are still some free places. If there are questions, feel free to contact me. Hope to see some of you. uday
[ccp4bb] Crystal rotation or crystal oscillation or both?
Dear Crystallographers, May be this is a very stupid question- which terminology should we use for crystal data collection- Crystal Rotation or Crystal Oscillation or both? I apologise if this is already discussed! Best, Dipankar -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway
[ccp4bb] PhD position in crystallography/EM - Imperial College, London
Applications are invited for a PhD studentship co-supervised by Dr. Harry Low and Prof. Martin Buck at Imperial College, London (South Kensington campus), starting from October 2015. Using a hybrid methods approach consisting of X-ray crystallography, electron microscopy and cell biology, the successful candidate will investigate how members of the phage-shock pathway promote bacterial persistence phenotypes during infection and antibiotic resistance. Applications are encouraged from outstanding European/UK students with or expecting to obtain a degree (2.1 or higher) in Chemistry, Biochemistry, Physics, Biophysics or related disciplines. A relevant Masters degree is also required. Previous experience in a structural biology related pursuit is desirable but not essential. For more information: http://www3.imperial.ac.uk/lifesciences/research/opportunities/studentships For informal enquiries please contact Dr. Harry Low (h@imperial.ac.ukmailto:h@imperial.ac.uk) or Prof. Martin Buck (m.b...@imperial.ac.ukmailto:m.b...@imperial.ac.uk) The deadline for applications is 30th April 2015. __ Dr. Harry Low Wellcome Trust Research Fellow Department of Life Sciences 512 Sir Ernst Chain Building Imperial College London, SW7 2AZ +44 207 594 3064
Re: [ccp4bb] adjusting bad Ramachandran angles
To give a short practical rather than a lengthy philosophical answer: - in real space: you can try Ramachandran restraints or secondary structure restraints -- indeed, both can be switched on in Coot in the refinement and regularisation parameters window (R/RC button in the top right corner). Or can try prosmart restraints as mentioned by others. And for the super-lazy: Extensions--All Molecule--Refine/Improve Ramachandran Plot. - in reciprocal space: try restraints to a higher res reference structure that has better quality, e.g. prosmart for refmac or reference coordinates in buster. (But if you are indeed after a philosophical answer: with all due respect to those who believe in the sanctity of the Ramachandran plot as a validation tool, it seems that times are changing and many crystallographers now prefer better quality structures over pedantic validation tools… The Ramachandran plot is one tool in the validation toolbox and should be used as such, i.e. in the context of many other quality criteria, such as backbone geometry, rotamers, clashes, density fit etc, some of which are restrained or are refinement parameters anyway.) From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Michael Murphy Sent: 25 February 2015 17:09 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] adjusting bad Ramachandran angles Does anyone know of a way to adjust Ramachandran angles so that they fall within the preferred range? Either in Coot or possibly some online server? I have been trying to do it manually without much success, I was wondering whether there might another way to do it. -Thanks 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] Crystal rotation or crystal oscillation or both?
Hi The Rotation Method in Crystallography ed Arndt Wonacott uses both crystal oscillation and crystal rotation. However, most of the time these days we don't oscillate the crystal when we're working at synchrotrons, but occasionally do when working in the lab. On 26 Feb 2015, at 09:34, Dipankar Manna wrote: Dear Crystallographers, May be this is a very stupid question- which terminology should we use for crystal data collection- Crystal Rotation or Crystal Oscillation or both? I apologise if this is already discussed! Best, Dipankar -- Dipankar Manna Research Scholar Department of Chemistry University of Oslo Oslo, Norway Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH Chairman of International Union of Crystallography Commission on Crystallographic Computing Chairman of European Crystallographic Association SIG9 (Crystallographic Computing)
Re: [ccp4bb] adjusting bad Ramachandran angles
Hi, Sorry, can’t take the credit for that! The names that come to mind are Gerard Kleywegt and Alwyn Jones. I’ve actually said in the past that, the more criteria you add to optimising your model, the less likely that you can satisfy all the criteria and still have a wrong model! But I agree with the point I think Gert was making, which is that the Ramachandran plot doesn’t lend itself to a simple gradient-driven optimisation strategy, i.e. the correct answer isn’t likely to simply be downhill in a potential function that includes some kind of Ramachandran restraints, because you will almost certainly have to do something more exhaustive that will involve jumping local barriers. Randy - 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 RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk On 26 Feb 2015, at 04:37, Jeremy Tame jt...@tsurumi.yokohama-cu.ac.jp wrote: I think Goodhart's Law applies here (see the Wikipedia page): When a measure becomes a target, it ceases to be a good measure. From memory I believe Randy Read and George Sheldrick have commented that Ramachandran plots are a good measure of structure quality, and therefore should not be used explicitly at the modelling stage. Some residues may be difficult if they have more than one backbone conformation or are just mobile, but expressly holding them in favoured regions of the Ramachandran plot is not a good idea. The most interesting proteins are of course enzymes, and the Ramachandran outliers are often among the most interesting active site residues. So you may be trying to eliminate something which is actually more important than a low Rfactor. The idea of limiting data use may seem counter-intuitive, but to take another example from economics, John Cowperthwaite was in charge of Hong Kong's financial affairs in the 1960s. He attributed the success of the economy under his tenure to his adamant refusal to collect any economic data whatsoever! On Feb 26, 2015, at 2:08 AM, Michael Murphy wrote: Does anyone know of a way to adjust Ramachandran angles so that they fall within the preferred range? Either in Coot or possibly some online server? I have been trying to do it manually without much success, I was wondering whether there might another way to do it. -Thanks
[ccp4bb] Ramachandran plot
I saw several excellent remarks about Ramachandran plots come by, but the main points still seem missing, I think. Phi and psi are not fixed parameters with limited freedom like angles, bond lengths or planarities. By keeping a certain bond lengths restrained at, for example, 1.543+/-0.021 Angstrom, you know what you are doing, and you know that exceptions are very, very rare (assuming that the weight on this restraint is set appropriately). Phi and psi, though, can have very many values. And especially near beta-bulges or near the ends of helices (or in active sites) one can easily do great damage to the realism of the coordinates if phi-psi restraints would be used in accordance with the local secondary structure. The variability of a bond length or bond angle is determined by simple physical, mainly local parameters such as through-bond and through-space forces acting on the direct neighbour atoms, and this happens in a way that we understand and that we can model in a coherent fashion. Phi and psi angles, similarly, are greatly influenced by interaction of the four atoms that determine each torsion angle with atoms in their direct local environment. Robbie explained this nicely in his message. These forces, though, are already restrained by the bond length, bond angle, and clash avoidance restraints. If you now put in additional restraints for phi and psi, you start counting certain interactions double in the restraints. Unless you spend a lot of time on re-calibrating all restraints, you are likely to generate areas where the sum of all restraints is too high and areas where not enough restraints are left. Again, I am not a crystallographer, but I am sure that that must make R-free go up. If using phi-psi restraints would make structures geometrically better while at the same time make R and R-free go down, than we should surely stimulate phi-psi restraints, but till this is proven, we shouldn't use them. If one day the community concludes that phi-psi constraints are a good thing, then please don't worry about the validation aspects. I am sure we will come-up with novel validation methods. Gert Het Radboudumc staat geregistreerd bij de Kamer van Koophandel in het handelsregister onder nummer 41055629. The Radboud university medical center is listed in the Commercial Register of the Chamber of Commerce under file number 41055629.
Re: [ccp4bb] Skin on drops
Hello Ulrike, In addition to other answers, one of the more esoteric (but surprisingly effective) ways to destroy the protein 'skin' on drops is to add a tiny bit of Trypsin solution. Crystals generally tend not to be affected, but (at least in my experience) the protein skin dissolves within a couple of hours or less. In at least one case, this was the only way to get rid of skin, which otherwise tugged on crystals and resulted in considerably worse diffraction. Good luck, Artem P.S. needless to say, if your skin is made of something else then trypsinolysis won't work :) - Cosmic Cats approve of this message On Wed, Feb 25, 2015 at 2:34 AM, Ulrike Demmer ulrike.dem...@biophys.mpg.de wrote: Dear crystallographers, I am trying to crystallize a soluble protein which tends to form aggregates. The crystallization condition is 20% PEG 3350 + 0,2 M Na-Formate. During the crstallization process a thick skin is formed on top of the sitting-drops. As well the crystals are buried in precipitate. Before I start harvesting I try to remove the skin but still it is hardly possible to get any crystals out of these drops. Any suggestions how to avoid the formation of skin on crystallization drops ? Cheers, Ulrike
Re: [ccp4bb] different Kd and Km value
On Feb 26, 2015, at 4:51 PM, Srivastava, Dhiraj dhiraj-srivast...@uiowa.edu wrote: Hi I have a protein with two substrate. when I am doing the binding studies with the two substrate separately, I am finding one of the substrate to have similar kd and Km. however the km and kd values are almost 30 fold different for the other substrate. it binds 30 fold more tightly then you can think based on Km. I don't know how to explain this. Km and Kd can be different but does any one have seen that much difference in Kd and Km? Thank you Dhiraj If k+2 isn’t small compared to k-1, Km cannot be approximated by Kd.
[ccp4bb] Fwd: [ccp4bb] adjusting bad Ramachandran angles
Dear Kevin Please re-read my earlier email paying particular attention to the words explicitly and expressly. To my mind, the notion of moving a Ramachandran outlier by moving a dot across a graph (which you feel is a danger) is identical to expressly forcing that residue into a favoured Ramachandran region. What you suggest doing is pretty much what I do do. I pay a great deal of attention to the Ramachandran plot at every stage of refinement, noting outliers and looking for the sorts of problems you mention, but I do not attempt to correct register errors (for example) by restraining phi/psi angles! Gert and Robbie have explained clearly why phi/psi angles do not lend themselves easily to fitting, and it seems best (to me at least) to leave the Ramachandran plot as an unfudged indicator of the interesting/problematical residues, not only for subsequent PDB users but for the crystallographer him/herself during refinement. I think we are in perfect agreement up to this point. Where we differ is in your apparent view that an outlier residue must be fixed. The first structure I ever solved had one residue with unusual backbone geometry, and I was given substantial grief from a modeller in the group over it - but it turned out to be a crucial functional residue at a hinge which flips between open and closed forms. Of course, it can be a pain explaining to a reviewer/editor/supervisor/PDB-bod why your model is flagged by validation software, but sometimes this is where the interesting bits are. If we have such a strongly defined preconception of what proteins look like then isn't the experimental data becoming undervalued? There are many parts of proteins that are not modelled well by single conformations, and the expectation that every atom will sit nicely in one place and behave is sometimes a little unrealistic. Dear Judit I am sure there are cases at AstraZeneca where repeated refinement of a well-known protein with umpteen different test drugs is expedited by the tools you suggest, but the aim of such experiments is rather different from an initial structure determination. I suspect that if Michael Murphy had wanted to know how to use a prior high-resolution model as a set of restraints for later lower resolution refinements then he would have framed his original question rather differently. When you say many crystallographers now prefer better quality structures over pedantic validation tools... I have to wonder what is meant by quality, but that is perhaps opening too large a can of worms. Basically though, beauty and truth are not synonymous, whether or not we regard protein crystallography as a video game where the person with the lowest R-factor wins. best wishes to all! Jeremy the Pedant Begin forwarded message: From: Kevin Jude kj...@stanford.edu Date: February 27, 2015 3:25:24 AM GMT+09:00 To: Jeremy Tame jt...@tsurumi.yokohama-cu.ac.jp Cc: CCP4BB@jiscmail.ac.uk Subject: Re: [ccp4bb] adjusting bad Ramachandran angles I think the Ramachandranplot should be used in the refinement and rebuilding process - a Ramachandran outlier is a flag that that region of the model needs a closer look, and the fix may be more complicated than simply rotating a peptide. Maybe a C-beta is pointing the wrong way, maybe there is a register error. The danger is that people will treat a Ramachandran outlier by moving a dot across a graph without addressing the underlying structural problem. kmj On Wed, Feb 25, 2015 at 8:37 PM, Jeremy Tame jt...@tsurumi.yokohama-cu.ac.jp wrote: I think Goodhart's Law applies here (see the Wikipedia page): When a measure becomes a target, it ceases to be a good measure. From memory I believe Randy Read and George Sheldrick have commented that Ramachandran plots are a good measure of structure quality, and therefore should not be used explicitly at the modelling stage. Some residues may be difficult if they have more than one backbone conformation or are just mobile, but expressly holding them in favoured regions of the Ramachandran plot is not a good idea. The most interesting proteins are of course enzymes, and the Ramachandran outliers are often among the most interesting active site residues. So you may be trying to eliminate something which is actually more important than a low Rfactor. The idea of limiting data use may seem counter-intuitive, but to take another example from economics, John Cowperthwaite was in charge of Hong Kong's financial affairs in the 1960s. He attributed the success of the economy under his tenure to his adamant refusal to collect any economic data whatsoever! On Feb 26, 2015, at 2:08 AM, Michael Murphy wrote: Does anyone know of a way to adjust Ramachandran angles so that they fall within the preferred range? Either in Coot or possibly some online server? I have been trying to do it manually without much success, I was wondering whether
