Re: [ccp4bb] delete subject
Dear Tom, don't feel too bad about it - everyone can make a mistake. Some of the replies give crystallographic tips that may be useful to other beginning and not-so-beginning crystallographers. Although I agree the attachments to the first mail would perhaps better be deleted from the records. Greetings, Mark Quoting Tom Van den Bergh : Is it possible to delete my post: refinement protein structure from ccp4 bb, i get too many bad reactions. I think its bettter to just delete the whole topic. Greetings, Tom
Re: [ccp4bb] refining against weak data and Table I stats
perhaps a second table in which certain statistics (Rsym, I/sigma, CC0.5) are given as a function of, say, 10 bins of resolution would be more useful than the same table twice at different resolution cutoffs. then editors, reviewers and ultimately readers can decide for themselves what resolution to call your structure. completeness and multiplicity could be included also in this table if they vary significantly with resolution (i.e. data in corners of square detectors) Quoting Robbie Joosten: Hi Douglas, Using two Table Is is a good way to show the difference between the two cut-offs, but I assume you will only discuss one of the models in your paper. IMO you only need to deposit the high res model, so there should be no problems with resolution conflicts in the PDB file. The annotators will probably help you if there is a problem with Rmerge > 1.00. As for the title of your paper: nobody forces you to put a resolution in it if it causes to much of a stir. Cheers, Robbie -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Boaz Shaanan Sent: Friday, December 07, 2012 12:21 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] refining against weak data and Table I stats Hi, I'm sure Kay will have something to say about this but I think the idea of the K & K paper was to introduce new (more objective) standards for deciding on the resolution, so I don't see why another table is needed. Cheers, Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220 Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Douglas Theobald [dtheob...@brandeis.edu] Sent: Friday, December 07, 2012 1:05 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] refining against weak data and Table I stats Hello all, I've followed with interest the discussions here about how we should be refining against weak data, e.g. data with I/sigI << 2 (perhaps using all bins that have a "significant" CC1/2 per Karplus and Diederichs 2012). This all makes statistical sense to me, but now I am wondering how I should report data and model stats in Table I. Here's what I've come up with: report two Table I's. For comparability to legacy structure stats, report a "classic" Table I, where I call the resolution whatever bin I/sigI=2. Use that as my "high res" bin, with high res bin stats reported in parentheses after global stats. Then have another Table (maybe Table I* in supplementary material?) where I report stats for the whole dataset, including the weak data I used in refinement. In both tables report CC1/2 and Rmeas. This way, I don't redefine the (mostly) conventional usage of "resolution", my Table I can be compared to precedent, I report stats for all the data and for the model against all data, and I take advantage of the information in the weak data during refinement. Thoughts? Douglas ^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^`^` Douglas L. Theobald Assistant Professor Department of Biochemistry Brandeis University Waltham, MA 02454-9110 dtheob...@brandeis.edu http://theobald.brandeis.edu/ ^\ /` /^. / /\ / / /`/ / . /` / / ' ' ' Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Strange density
could it be a PEG molecule? Quoting "Read, Jon": Anyone see anything like this before? The data is 1.7Angstrom data with good statistics. The picture shows the solid FoFc density contoured at 3 Sigma in light brown and -3 Sigma in purple. The density is odd as it appears to be bound to a peptide carbonyl with no other obvious interactions with the protein. There is a characteristic tail at one end. -- 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. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] ccp4mg question
possible ugly workaround: renumber the pdb file so where ccp4mg thinks the N and C-termini are is hidden in the image you are making (if possible) if you want to make different views you may need to make differently renumbered pdb files. (but probably other people have a smarter way) as an aside, PyMol I think does not have these problems, at least we make images of cyclic peptides with it and I haven't run into it. Quoting "SANCHEZ BARRENA, MARIA JOSE": Dear all, I am working with a cyclic protein and I am trying to make a figure with ccp4mg. I would like to know how to say to ccp4mg that the N and C-terminus are bound Although atoms are at a covalent bond distance, the chain is broken by ccp4mg... Many thanks in advance for your suggestions and help! Maria Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] side chain density
before modelling a long side-chain in non-existing or dubious density, also make sure it is really there in the protein by sequencing your expression plasmid. Your arginine (for example) may in fact be a serine or glycine...databases are not 100% accurate and neither is PCR if it was used in the cloning. Quoting Ed Pozharski: OK, here we go again. This has been argued ad nauseam, see for example http://www.dl.ac.uk/list-archive-public/ccp4bb/msg19738.html or http://www.mail-archive.com/ccp4bb@jiscmail.ac.uk/msg20268.html (hard to believe we have gone more than a year without another version of "what to do with disordered side chains" 250-post long discussion :) I do not have much to add to the above, however On 11/09/2012 05:22 PM, Matthew Franklin wrote: I think we can all agree that virtually every structure in the PDB will have a few residues where some of the atoms are not visible in the density. So the "trim the side chains" crowd is a well-represented minority at 30%, but 70% of depositors chose another option. This maybe the historical average, I suspect that currently the "trim the side chains crowd" may be at least at 50% (but what about Ohio? :). Majority, however, is not always right (don't get me started on I-over-sigma ratios). I personally like to leave all atoms on the side chains; they look wrong to me when beheaded. I just try to put the invisible atoms in a stereochemically plausible conformation, leave the occupancy set to 1, and let the refinement program deal with them. With all due respect, to model something where there is no density (aka experimental evidence) cannot be justified by aesthetics. On the contrary, there is some evidence suggesting that modelling disordered side chains in the way you describe adds small, but detectable error to the rest of the model. Cheers, Ed. -- Oh, suddenly throwing a giraffe into a volcano to make water is crazy? Julian, King of Lemurs Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] protein cleavage
We once had a more-or-less MBP-sized fragment before cleavage, but this turned to be a spontaneous mutation. This expression experiment had been started from a glycerol stock with an unknown number of growth cycles prior to expression. Starting from a fresh transformation with the purified and sequenced plasmid solved the problem. Since then, I insist everybody does a fresh transformation before every expression experiment and not generate extra growth/dilution cycles beyond the normal transformation, growth on plate, overnight culture, dilution into large-scale expression culture. Quoting "Bosch, Juergen": @Cynthia, On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote: Since you're seeing the MBP band, it sounds as if you're getting some cleavage. If there were no cleave you would only see the fusion and TEV bands on the gel. I think that is a wrong assumption. He did not specify if he sees the MBP band also just before TEV addition - it might also be truncation products which we happen to see all the time. The ratio varies depending on the construct but it can be as bad as a 1:1 ratio. You can really only tell if TEV cleaves if you do a time course experiment at RT with a defined amount of your protein and see if the fusion construct decreases. An alternative for the lack of your 17kDa desired band is simply your fusion construct is cleaved but your cleaved product might a) not be soluble at that pH or b) aggregates and precipitates. You might be able to perform the cleavage on the Amylose column keeping a constant flow cycling. Jürgen .. Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Plate crystals
apart from optimising the crystallisation conditions, it might be worth optimising the protein preparation or do some limited proteolysis - or even express short N-terminal and/or C-terminal deletions. Mark Quoting Patrick Shaw Stewart: Jahan It sounds as though the protein crystallizes well, so microseeding (done the right way) is very likely to solve the problem. Make a strong seed stock with as much crystalline material as possible from several wells, including different hits if possible. Just mix them all together, but keep PEG conditions separate from salt conditions (or you will get two layers). Make a set of serial dilutions from "neat" up to 1 in 100,000 and freeze them at -80. You need to seed into *random screens*, so that you can pick up new conditions. Then you should optimize two or three new conditions by using the diluted seed stock. For example, if you estimate that there are 1000 crystals in the drop, you use the 1:1000 dilution. For info and references see http://www.douglas.co.uk/mms.htm On 15 October 2012 23:01, Jahan Alikhajeh wrote: Dear Friends, I am trying to crystalize a 70 kDa nasty protein but I got plate shape crystals with high mosaicity and useless diffraction (up to 4A). I tried to improve/optimize crystallization but either I got the same or nothing. I tried seeding but I had so many crystals without any improvement. Does anyone have better idea than routine optimization method in the lab? Thanks in advance. Jahan -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- patr...@douglas.co.ukDouglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] how to interpret DALI search results
...I meant visualisation software of course... Quoting "VAN RAAIJ , MARK JOHAN": if this is the first (or second, or third) time you do a DALI search, take the list output from DALI, start from the top and superpose each structure with yours and look at the superpositions with your favourite superposition software. This is very educational and the only way you get a feeling for what numbers mean. Quoting Jerry McCully: Dear ALL; After we solved our structure by anomalous scattering, we did a DALI search. Here are the results but it is not easy to draw meaningful conclusions whether our structure represents a novel fold or is homologous to others. Basically the Z-score is between 2 and 6.4 since our structure only contains 130 residues. Sequence identity is between 5 to 15%. The RMSD of structural alignment is between 2.5 to 6 angstrom. Any suggests to interpret the DALI results? Many thanks, Jerry McCully Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] how to interpret DALI search results
if this is the first (or second, or third) time you do a DALI search, take the list output from DALI, start from the top and superpose each structure with yours and look at the superpositions with your favourite superposition software. This is very educational and the only way you get a feeling for what numbers mean. Quoting Jerry McCully: Dear ALL; After we solved our structure by anomalous scattering, we did a DALI search. Here are the results but it is not easy to draw meaningful conclusions whether our structure represents a novel fold or is homologous to others. Basically the Z-score is between 2 and 6.4 since our structure only contains 130 residues. Sequence identity is between 5 to 15%. The RMSD of structural alignment is between 2.5 to 6 angstrom. Any suggests to interpret the DALI results? Many thanks, Jerry McCully Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Off-topic: PDB deposition of multiple structure factor files
so perhaps the problem indeed is sending different wavelengths as one file...in our case there is only one crystal, one wavelength, i.e. one loop, while I clearly submitted all three wavelengths. Quoting "Miller, Mitchell D.": We (JCSG) too have been depositing multiple data sets (including unmerged original index intensities for each wavelength and even for multiple crystals when one was used for phasing and another for refinement, and MAD phases and DM modified map coefficients) since 2004 without problems. These are all in separate data loops of a single structure factor file. Regards, Mitch -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jens Kaiser Sent: Friday, April 27, 2012 11:54 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off-topic: PDB deposition of multiple structure factor files It might be a portal issue. But the pdb staff is very helpful in getting this deposited. We deposited data of I think 4 "crystals" and 3 wavelengths with different phase sets in 2008. (The data was anisotropic, 3.5/4.2 A resolution, model building was not straight forward, so we wanted to preserve as much information as possible. If memory serves right, we have experimental fobs, anisotropy corrected fobs, a derivative and a semet dataset; if you're interested, pdb code is 3dhw, have a look at the sf-file) hth, Jens On Fri, 2012-04-27 at 20:35 +0200, Mark J van Raaij wrote: again, it looks like this is particular to the US portal. We submit via the European www.pdbe.org and can submit multiple datasets. See 2XGF for an example. Note: I think from www.rcsb.org only one file can be downloaded, but www.pdbe.org clearly shows both. Although you are in the US, you can use the pdbe deposition tool AUTODEP - or the Japanese one, if you like. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3 E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/~mjvanraaij On 27 Apr 2012, at 20:23, Florian Schmitzberger wrote: > Dear All, > > With my most recent PDBe deposition, in addition to the native data, I had intended to deposit the anomalous data, used for structure determination, and make it available for download. This turned out to be less straightforward than I had anticipated, because the current PDB convention is to only allow a single structure factor file for experimental data (usually the native dataset), available for download from the PDB. In my case, the anomalous data were concatenated with the native data into a single cif file (this worked and made sense, because both for both datasets the unit cell dimensions are virtually identical). > > I imagine it would be beneficial to be able to make available more than a single structure factor file, including the ones derived from experimental phasing, in the PDB, along with the final coordinates, without concatenating the data into a single file (which may lead to confusion to users when downloaded). Is this anything the PDB is already working to implement in the near future (perhaps via the coming PDBx format)? > > Best regards, > > Florian > > > > > > > > > > > > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] PEG MME 2000 in powder form?
ok, I stand corrected, Sigma DOES sell it, but under a slightly different name: 81321 FLUKA Poly(ethylene glycol) methyl ether average Mw 2,000 81321-250G, 23.60 euros 81321-1KG, 71.80 euros (prices given for Spain) thanks! Quoting "VAN RAAIJ , MARK JOHAN": > > > Dear All, > > is PEG MME 2000 still available in powder form? I think Fluka used to > sell it, but Fluka is no more and Sigma-Aldrich don't sell it. > > Hampton Research and Molecular Dimensions (and perhaps others) do > sell 50% (w/v) solutions. > > Mark > > Mark J van Raaij > Laboratorio M-4 > Dpto de Estructura de Macromoléculas > Centro Nacional de Biotecnología - CSIC > c/Darwin 3, Campus Cantoblanco > 28049 Madrid > tel. 91 585 4616 > email: mjvanra...@cnb.csic.es Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
[ccp4bb] PEG MME 2000 in powder form?
Dear All, is PEG MME 2000 still available in powder form? I think Fluka used to sell it, but Fluka is no more and Sigma-Aldrich don't sell it. Hampton Research and Molecular Dimensions (and perhaps others) do sell 50% (w/v) solutions. Mark Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] negative difference density around sulphur and oxygen atoms
apart from radation damage it could be a combination of: - too tight restraints on the B-factors - 9 sigma not being that much on a the e/A3 scale, i.e. your difference map is very flat (which is good) and the few peaks that remain stand out a lot, even if their absolute height is low... Quoting Chris Meier: Message Dear all, I am refining the X-ray structure of a protein: Data to ~2A were collected at a latest-generation synchrotron. The 2fo-Fc maps are crisp, the model of the protein is complete and I am reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac 5.5). However, I am seeing a lot of negative difference density, especially around sulphur atoms (negative density around -9 sigma) and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with negative density around -6 sigma). Has anyone observed this before? I have found CCP4bb postings discussing radiation damange of suplphur atoms (e.g. http://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html ). Can this also happen with oxygen atoms? What would be an appropriate way to deal with this issue during refinement? Suggestions greatly appreciated. Thanks, Chris Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] one datum many data?
and using "dice" for the singular "die" Quoting Robert Blessing: How can "spectrum" and "spectra" have been overlooked in this thread? Síocháin! Sláinte! In Irish: Peace! Health! Pronounced roughly "Shee'-kahn", "Slawn'-tche". Bob Robert H. Blessing, Ph.D. Senior Research Scientist Hauptman-Woodward Medical Research Institute, Inc. Professor of Structural Biology and Research Professor of Chemistry Director of Graduate Studies in Structural Biology Interim Chairman of the Department of Structural Biology State University of New York at Buffalo Hauptman-Woodward Institute 700 Ellicott Street Buffalo, New York 14203, USA Phone 716-898-8613 Fax716-898-8660 eMail bless...@hwi.buffalo.edu Internet http://www.hwi.buffalo.edu Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] one datum many data? [was Re: [ccp4bb] very informative - Trends in Data Fabrication]
another singular/plural grump: Recently we can read: "phage are". Phage is singular, the plural is phages (and this does not have that much to do with latin or greek). more reading: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3109450/ Quoting Paul Emsley: The PDBe page for 3k78 says: "The experimental data has been deposited" the data cif file says: "data is under question" Grump. Is it to late to refer to data as if there were more than one of them? Anyway, the data mtz file is here if you want to refine with it: http://lmb.bioch.ox.ac.uk/emsley/data/r3k78sf.mtz Paul. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] How to reduce no. of overlaps
typo correction, you'll want the long axis parallel to the rotation axis, not to the beam. Mark Quoting Frank von Delft: You probably have to tilt your crystal, so that the long axis is parallel to the beam. We do this routinely: cut a plastic pipette tip to have a sharp point, then push the loop where it attaches to the pin, to bend the crystal itself. You have to identify from your diffraction whether the long axis is pointing through the face or the edge of the loop. As it's P6, chances are it's through the face, because long-axis P6 tends to make flat hexagons which lie flush with the face. So you have to bend so the face of the loop upwards. You'll have to practice this first, though, so put up an empty loop. Top tips: * Don't breathe! You'll blow the cryostream away. * Bend the loop towards (rather than away from) the rim edge of the pin to which it's glued. * Don't breathe! * Practise practise practise. Another thing: most in-house sources allow you to reduce divergence of the beam. You lose intensity, but no matter, just expose longer. That also improves overlap. Cheers phx On 07/03/2012 04:56, Dipankar Manna wrote: Dear Crystallographers, I am working on a protein having SG P6, the cell parameters are a= 79, b= 79, c= 325. The crystals are forming in big size and with very good shape. It also diffracting very well in Home source facility both in terms of resolution and intensity. But the only problem is the number of overlaps. Its showing much more than the good spots. As a result the completeness is showing maximum up to 65% even after collecting 180 degrees. I am unable to get a complete data. I tried with reducing the oscillation angel to 0.3 degree/0.5 degree but it did not improve that much. Please give me some suggestions. Regards, Dipankar /Dipankar Manna/ */Aurigene Discovery Technologies Limited,/* /#39-40 KIADB Industrial Area, Electronic City, Phase II,/ /Hosur Road, Bangalore- 560 100, India/ /Cell: +91-9538631469 // | Office Ph : +91 80-66204422 (Extn: 398) | Email ID: dipanka...@aurigene.com/ This e-mail and any files transmitted with it are for the sole use of the intended recipient(s) and may contain confidential and privileged information.If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message.Any unauthorized review, use, disclosure, dissemination, forwarding,printing or copying of this email or any action taken in reliance on this e-mail is strictly prohibited and may be unlawful. Visit us at http://www.aurigene.com Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] pH optimisation for crystallisation
Dear Sreetama, First of all, there are no hard-and-fast "rules" for successful crystallisation, try changing as many different variables as possible and go with what works. Having said that, yes, next I would go for a grid optimisation varying the pH in 0.2 or 0.5 units over as wide a range as the buffer can reasonable tolerate at the same molarity, and try different precipitant concentrations on the other axis. If you have enough protein, try plates at different temperatures as well, and different protein concentrations (in multidrop wells you can do this in the same experiment). A very important variable is the protein preparation itself, prepare more protein regularly and try to improve on purity and concentration. Mark Quoting sreetama das: Dear all, I have a 17 KDa protein that gives crystals in a condition that has 0.1M bis-tris pH 6.5. The crystals are thin needle clusters and do not diffract. I have tried additives, but they haven't improved the crystals. I intend to vary the pH of the condition. My questions are- 1. should the buffer be kept the same or can it also be changed (as long as the desired pH is within the range of both the buffers)? 2. in case of a different buffer, should its molarity be the same as that of the original one in the crystallization condition? regards, sreetama Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] symmetry for ages 6 and up
(oops, previous mail got sent before I wanted) the turtles would be really nice to extend things into 3D. great find, Mark Quoting Phoebe Rice: Hi all, For those who teach xtallography - we found some plastic turtles that can be snapped together in an amazing variety of space groups. Worked well in a workshop for our students, so I thought I'd share the shopping tip. They're called Reptangles, and we got them from Amazon. http://www.amazon.com/Fat-Brain-Toys-FA042-1-Reptangles/dp/B00392NSQ4 Have fun! Phoebe = Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] symmetry for ages 6 and up
reminds me of these symmetric 2D P3 lizards: http://www.worldofescher.com/store/Z51.html I bought the RGB-coloured set/puzzle after visiting an Escher exhibition and sometimes use them in crystallography/symmetry teaching. Nice to make the students assemble them and then decide on the symmetry operator, unit cell and asymmetric unit. Quoting Phoebe Rice: Hi all, For those who teach xtallography - we found some plastic turtles that can be snapped together in an amazing variety of space groups. Worked well in a workshop for our students, so I thought I'd share the shopping tip. They're called Reptangles, and we got them from Amazon. http://www.amazon.com/Fat-Brain-Toys-FA042-1-Reptangles/dp/B00392NSQ4 Have fun! Phoebe = Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago phone 773 834 1723 http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 http://www.rsc.org/shop/books/2008/9780854042722.asp Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] SeMET inconsistency
Dear Kavya, I don't think it is likely you have five MSE and one MET. Rather, I would guess the side-chain has some disorder, i.e. one or more alternative conformations. If you don't see density for alternative conformations, the best way to model the disorder might be partial occupancy of the MSE that gives negative difference density peaks. Mark Quoting Kayashree M: Dear users, I had posted this question already but in a different context. One of the Se-Met derivatised protein that we have solve is a homodimer (with 4 MET in the chains that crystallised) of which one chain has 3 MSE residues, while the other chain has only 2 MSE. Are there any such instances in PDB, where two homodimer (or any mer) wherein each has different percentage of MSE? Because when we change the only MET to MSE a negative density would arise. The tools to analyse anomalous peaks is not giving peaks for the MSE residues as the data was collected at 1.541Ang wavelength. Thank you Kavya -- This message has been scanned for viruses and dangerous content byMAILSCANNER[1], and is believed to be clean. Links: -- [1] http://www.mailscanner.info/ Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Complex seeding
agree, any crystallisation idea is worth pursuing, given you have or can make enough sample to try it with. having said that, wouldn't you tend to select for the same crystals as the seed, i.e. crystals of the component on its own? have you tried limited proteolysis of your sample, incl. a bit of protease in the drop - or can you think of ways to stabilise the complex? Mark Quoting Ed Pozharski: > On Wed, 2011-09-21 at 18:04 +0100, Peter Hsu wrote: >> Or is this just a crazy/bad idea? > > If there is one thing that I learned about crystallization, is that very > few ideas are so crazy that they are bad (i.e. not worth trying). Well, > if dried seaweed and ground horse hair are good for seeding, I don't see > how actual protein crystal seeds can be dismissed. > > > > -- > Oh, suddenly throwing a giraffe into a volcano to make water is crazy? > Julian, King of Lemurs > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Complex seeding
agree, any crystallisation idea is worth pursuing, given you have or can make enough sample to try it with. having said that, wouldn't you tend to select for the same crystals as the seed, i.e. crystals of the component on its own? have you tried limited proteolysis of your sample, incl. a bit of protease in the drop - or can you think of ways to stabilise the complex?Mark Quoting Ed Pozharski: > On Wed, 2011-09-21 at 18:04 +0100, Peter Hsu wrote: >> Or is this just a crazy/bad idea? > > If there is one thing that I learned about crystallization, is that very > few ideas are so crazy that they are bad (i.e. not worth trying). Well, > if dried seaweed and ground horse hair are good for seeding, I don't see > how actual protein crystal seeds can be dismissed. > > > > -- > Oh, suddenly throwing a giraffe into a volcano to make water is crazy? > Julian, King of Lemurs > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Data from old tapes
did you try looking if there is a company offering data recovery services from these kind of tapes? if there is, there may not be a need to buy a tape drive yourself.Mark Quoting "Min, Xiaoshan": > Dear CCP4 community, > > We have a few datasets that reside in tapes (Sony QG-112M 8 mm tape > and Maxell DDS-2 4 mm tape). I have been searching the internet for > tape drives ( and cable) but haven't found anything. Does anyone > know where we can purchase compatible tape drives for these lovely > tapes? Or if you have a spare working set sitting in your graphic > room and would like to sell them, that will be wonderful. Thanks. > > > Xiaoshan Min. Ph.D. > Molecular Structure > Amgen San Francisco > 1120 Veterans Blvd. > South San Francisco, CA 94080 > > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Off topic question. NACCESS
Dear Armando, don't know about NACCESS, but I guess it is superseded by AREAIMOL in CCP4 (also in CCP4i); it outputs the accessible volume per atom in the pdb file and per residue and per chain and some other statistics in the log-file. Mark Quoting Armando Albert: > Does anyone has got some information about how to get a mac version > (intel), of the old unix program naccess?. It was meant to calculate > the solvent accessibility per residue from a pdb file. > Armando Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] how to quantitate protein which dont have ne aromatic residue
you can do amino acid analysis on your pure protein, using a commercial or academic service - I hope these are still around. You should only need to do this once, then relate the result to your A220, BCA and Bradford assays. Mark Quoting Arpit Mishra: > hello everybody > > i am working on the protien which dont have any aromatic residue i do fplc > other purification using 220 absorption, but i want to quantitate protein > precisely i have tried using BCA nd bradford but both methods quantification > is not matching,,so any one is having sum idea how to quantitate it > precisely > > thanks in advance for your valuable suggestion.. > > > Arpit Mishra > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] OT: PCR instrument
if the PCR machine is just to be used for standard sub-cloning (amplifying fragments from other plasmids, cloned cDNA etc.), I would go for the cheapest one I could find. I guess there are few crystallography projects were the first PCR step turned out to be the most difficult. For more sophisticated applications there are probably forums where more knowledgeable people reside (on PCR that is...) Mark Quoting "Bernhard Rupp (Hofkristallrat a.D.)": > Dear All, > > I was polled for a recommendation for a good PCR instrument, > but I am not much of a molecular biology person - if someone could > please help and kindly send some recommendations to > > Eric W. Reinheimer > > Best regards, BR > - > Bernhard Rupp > 001 (925) 209-7429 > +43 (676) 571-0536 > b...@ruppweb.org > hofkristall...@gmail.com > http://www.ruppweb.org/ > - > No animals were hurt or killed during the > production of this email. > - > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] what to do with disordered side chains
yet, apart from (and additionally to) modelling two conformations of the side-chain, the B-factor is the only tool we have (now). Quoting Pavel Afonine: > Hi Quyen, > > > (...) And if B-factor is an estimate of thermo-motion (or static disorder), >> then would it not be reasonable to accept that building the side-chain and >> let B-factor sky rocket might reflect reality more so than not building it? >> > > NO. Your B-factors are valid within a harmonic (small) approximation of > atomic vibrations. Larger scale motions you are talking about go beyond the > harmonic approximation, and using the B-factor to model them is abusing the > corresponding mathematical model. > http://www.phenix-online.org/newsletter/CCN_2010_07.pdf > > Pavel. > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] off-topic: 2 peaks on Cation
I observed 3-5 peaks for a baculovirus expressed protein once, and could only get good crystals if the peaks were crystallised separately, see: Virology 262, 333-343 (1999) Virology 262, 333–343 (1999) Mark Quoting Ulli Hain: > Thanks for the suggestions/ideas. The protein is recombinantly > expressed in E.coli. It does in fact show a metal dependency. We mass > spec'd the peaks once looking for phosphorylation, which was not > detected, but we only got about 60-70% sequence coverage so it was > not very helpful. > > Quoting "Nadir T. Mrabet" : > >> Given no info on the protein, it can be anything. >> Is it recombinant? Which host? etc. >> >> Oxydation (cys, met) is also a possibility >> By the way, deamination concerns asn and gln, not lys. >> >> Best, >> >> Nadir >> >> Pr. Nadir T. Mrabet >> Structural& Molecular Biochemistry >> Nutrigenex - INSERM U-954 >> Nancy University, School of Medicine >> 9, Avenue de la Foret de Haye, BP 184 >> 54505 Vandoeuvre-les-Nancy Cedex >> France >> Phone: +33 (0)3.83.68.32.73 >> Fax: +33 (0)3.83.68.32.79 >> E-mail: Nadir.Mrabet medecine.uhp-nancy.fr >> >> >> >> On 18/02/2011 19:45, Soisson, Stephen M wrote: >>> Possibly deamidation of the protein, in particluar one or more >>> lysines. What does the Mass spec look like? >>> Cheers, >>> Steve >>> >>> >>> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On >>> Behalf Of *Ulli Hain >>> *Sent:* Friday, February 18, 2011 12:14 PM >>> *To:* CCP4BB@JISCMAIL.AC.UK >>> *Subject:* [ccp4bb] off-topic: 2 peaks on Cation >>> >>> Hi, I was wondering if anyone had possible explanations for a >>> recombinantly expressed soluble protein that runs as 2 equal, >>> slightly overlapping peaks on a cation exhanger but as one peak on a >>> size exclusion column and same electrophoretic mobility on SDS-PAGE. >>> -Ulli >>> >>> >>> Adelaide Ulricke Hain >>> PhD Candidate >>> Johns Hopkins Bloomberg School of Public Health >>> Department of Biochemistry and Molecular Biology >>> 615 North Wolfe Street >>> Baltimore, MD 21205 >>> Notice: This e-mail message, together with any attachments, contains >>> information of Merck& Co., Inc. (One Merck Drive, Whitehouse Station, >>> New Jersey, USA 08889), and/or its affiliates Direct contact information >>> for affiliates is available at >>> http://www.merck.com/contact/contacts.html) that may be confidential, >>> proprietary copyrighted and/or legally privileged. It is intended solely >>> for the use of the individual or entity named on this message. If you are >>> not the intended recipient, and have received this message in error, >>> please notify us immediately by reply e-mail and then delete it from >>> your system. >> Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Problems with auto-indexing
Dear Chen, It looks like you have a unit cell with two relatively short axes and one long one - and some disorder. I have experienced a few examples of this with virus fibre proteins (adenovirus fibre and T4 short tail fibre). For the short tail fibre we also obtained images in which some lines of spots showed nice separation but others less so. You should try and mount the crystals with the long axis roughly parallel to the rotation axis to avoid overlaps. If you screen several (or many) crystals, you may find one with less disorder. In any case, collect complete datasets of the best ones and try to solve the structure, you may get lucky like we did. Greetings, Mark Quoting chen c: > I attached two diffraction images of my crystal, of which one seems normal > as protein crystal usually do, while the other one looks very strange ,with > very continuous lines on the image. > > In fact, of the 180 crystal images diffracted by my crystal, there is a > tendency between those two. > > I had thought that my crystal is a combination of many two-dimensional > crystals, between wich there are translational or rotational translocations, > namely resulting in a lack of translational symmetry in the third axes. As a > result of this, when I tried to index them using HKL2000, one of the cell > parameter is merely several angstroms. > > However, of the several data sets from different crystals, one data set is > sucessfully indexed by the assistant professor of my laboratory and > currently submitted to the operation of Molucular Replacement. > > This confused me a lot. Is what I thought wrong? Or is the very crystal that > was indexed a special one? > > Thanks all > > chen > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] relationship between B factors and Koff
Hi Sebastiano, I don't see how the k-off would influence this, given the timescale of growing crystals. An explanation in terms of high Kd and relative lack of crystal contacts for the component with higher temperature factors would sound more convincing to me. Mark Quoting Vellieux Frederic: > I can direct you to PDB entry 1EWY, where the average isotropic > temperature factor for the major component of the complex is ca. 47 > A**2 and that for the smaller component is ca. 69 A**2. Similar > values than the ones you are reporting. I am assuming some sort of > "disorder", or if you prefer, "wobbling" of the smaller component at > the lever of the binding site. > > Fred. > > Sebastiano Pasqualato wrote: >> Hi all, >> I have a crystallographical/biochemical problem, and maybe some of >> you guys can help me out. >> >> We have recently crystallized a protein:protein complex, whose Kd >> has been measured being ca. 10 uM (both by fluorescence polarization >> and surface plasmon resonance). >> Despite the 'decent' affinity, we couldn't purify an homogeneous >> complex in size exclusion chromatography, even mixing the protein at >> concentrations up to 80-100 uM each. >> We explained this behavior by assuming that extremely high Kon/Koff >> values combine to give this 10 uM affinity, and the high Koff value >> would account for the dissociation going on during size exclusion >> chromatography. We have partial evidence for this from the SPR >> curves, although we haven't actually measured the Kon/Koff values. >> >> We eventually managed to solve the crystal structure of the complex >> by mixing the two proteins (we had to add an excess of one of them >> to get good diffraction data). >> Once solved the structure (which makes perfect biological sense and >> has been validated), we get mean B factors for one of the component >> (the larger) much lower than those of the other component (the >> smaller one, which we had in excess). We're talking about 48 Å^2 vs. >> 75 Å^2. >> >> I was wondering if anybody has had some similar cases, or has any >> hint on the possible relationship it might (or might not) exist >> between high a Koff value and high B factors (a relationship we are >> tempted to draw). >> >> Thanks in advance, >> best regards, >> ciao >> s >> >> >> > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
Re: [ccp4bb] Editorial policies (was Citations in supplementary material)
Regarding editorial decisions, I actually welcome editors making more rejection decisions, i.e. reading the paper before sending it to referees, so I waste less time, waiting as author, or as referee reading and commenting on papers for which it would have been clear beforehand that they are not suitable for the journal. I realise this would put more burden on the editors, but then they could enlist more editor (I have never been asked to be editor for any journal yet (hint)...). Mark Quoting Anastassis Perrakis: > Dear John, > > I did not have the IUCr journals specifically in mind while making > these remarks. > Quite the contrary, I think due to you and other colleagues and > friends, they are > run competently and to the benefit of the community and of science at large. > > If I may though offer an opinion about the peer review system in IUCr > journals, > I personally find the concept that the authors know the identity of > the managing > editor, wrong. I am sure that in the majority of cases its not a problem, > but often a managing editor can hesitate to communicate a negative > referee report > to e.g. an old colleague or good friend, whose manuscript she/he is > handling, > even when one of the referees is negative. > > I much prefer the system of e.g. Proteins (where I act as a managing > editor), > where authors never learn the identity of the managing editor far > more comfortable > (there is a few people that I would rather prefer if they don't know > for sure that I rejected their paper), > and the current system of PNAS were you learn the identity of the > editor only if your paper is accepted and after is published, far > superior (you make friends but not enemies ...) > > I would not mind to had seen IUCr journals adopting a similar system, > I think it would improve even further > their good reputation. > > A. > > > On Nov 19, 2010, at 12:32, John R Helliwell wrote: > >> I don't wish to vear away from Victor's thrust with starting this >> thread and I would happily sign the petition you suggest. >> >> But I feel I should respond to the assertions about 'problems of peer >> review' at least with respect to Journals of my experience. >> Some 'Editor handling of submissions' statistics should help quantify >> such matters. These are a matter of public record re my IUCr Journals >> submission handling statistics ie therefore not confidential and which >> basically are:- >> approx 1000 article submissions; >> my rejection rate 20%; >> appeals against my rejections 0.5%; >> As Editor in Chief of Acta Cryst between 1996 to 2005 I received three >> appeals (out of approx tens of thousands of submissions through all >> Coeditors); I rejected these three. [My judgements were confidential >> re the details.] >> >> I can add that for the 2000 referees' reports or so for my article >> handling of submissions, that colleagues have kindly supplied to my >> Editor requests, problems involve:- >> about 1% where the report is 'publish as is' AND without any >> commendation given; these are in effect not terribly useful reports to >> me as an Editor. Another problem, which is growing, is the number of >> declines to my invites to referee (around 10%). Even worse are the no >> replies at all from invited referees as time is lost to the authors >> who rightly expect as prompt as possible handling. >> >> Re your points I offer replies as follows:- >> "Let me outline what I think are problems of peer review: >> >> 1. 'review by last author name'. Very often the last author is well >> known, or a friend, and the reviewers' critical judgement takes a >> temporary leave of abesnse. >> JRH reply:- Such reports would be easy to spot and are not a problem >> in my experience and so resort to double blind review is not necessary >> in my experience. >> >> 2. 'preferred reviewers'. a double edged sword .. think about it. >> JRH reply; these are not so commonly offered suggestions by authors in >> fact and where they are one can follow or decide against (see point >> 1). >> >> >> 3. too much power of decision on editors (professional or academic) >> being able to reject papers without peer-review in many journals. >> JRH reply;This approach, 'insufficent general interest' is for the >> magazines we know and yet still love. >> >> 4. Bad refereeing - sometimes I wonder if people read the paper. >> JRH reply;Such reports are very few and obvious. The other categories >> above are more common (ie 'publish as is' category). >> >> 5. Lack of referee expertise: you get papers these days with: a >> structure, some biochemistry, some SAXS, some biophysics, and a cell >> based assay. Two or three people being >> able to pick up all the mistakes is very unlikely. >> JRH reply; Papers can be challenging re content and your example here >> is a good one. Other chalenging cases are where they include a lot of >> maths. That said peer review does its best but can occasionally fail; >> this level of f
Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
perhaps we should campaign for it to be obligatory to provide the pdb and structure factor file to the journal, and thus referees, upon submission? Then he can look for himself to see that building and refinement have been performed satisfactorily. Mark > Surely the "best" model is the one that the referees for your paper > are happy with? > > I have found referees to impose seemingly random and arbitrary > standards that sometime require a lot of effort to comply with but > result in little to no impact on the biology being described. Mind > you discussions on this email list can be a useful resource for > telling referee's why you don't think you should comply with their > "rule of thumb". > > Simon > > > > On 27 Oct 2010, at 20:11, Bernhard Rupp (Hofkristallrat a.D.) wrote: > >> Dear Young and Impressionable readers: >> >> I second-guess here that Robbie's intent - after re-refining many many PDB >> structures, seeing dreadful things, and becoming a hardened cynic - is to >> provoke more discussion in order to put in perspective - if not debunk- >> almost all of these rules. >> >> So it may be better to pretend you have never heard of these rules. Your >> crystallographic life might be a happier and less biased one. >> >> If you follow this simple procedure (not a rule) >> >> The model that fits the primary evidence (minimally biased electron density) >> best and is at the same time physically meaningful, is the best model, i. >> e., all plausibly accountable electron density (and not more) is modeled. >> >> This process of course does require a little work (like looking through all >> of the model, not just the interesting parts, and thinking what makes sense) >> but may lead to additional and unexpected insights. And in almost all cases, >> you will get a model with plausible statistics, without any reliance on >> rules. >> >> For some decisions regarding global parameterizations you have to apply more >> sophisticated test such as Ethan pointed out (HR tests) or Ian uses >> (LL-tests). And once you know how to do that, you do not need any rules of >> thumb anyhow. >> >> So I opt for a formal burial of these rules of thumb and a toast to evidence >> and plausibility. >> >> And, as Gerard B said in other words so nicely: >> >> Si tacuisses, philosophus mansisses. >> >> BR >> >> -Original Message- >> From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Robbie >> Joosten >> Sent: Tuesday, October 26, 2010 10:29 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) >> >> Dear Anthony, >> >> That is an excellent question! I believe there are quite a lot of 'rules of >> thumb' going around. Some of them seem to lead to very dogmatic thinking and >> have caused (refereeing) trouble for good structures and lack of trouble for >> bad structures. A lot of them were discussed at the CCP4BB so it may be nice >> to try to list them all. >> >> >> Rule 1: If Rwork < 20%, you are done. >> Rule 2: If R-free - Rwork > 5%, your structure is wrong. >> Rule 3: At resolution X, the bond length rmsd should be < than Y (What is >> the rmsd thing people keep talking about?) Rule 4: If your resolution is >> lower than X, you should not use_anisotropic_Bs/riding_hydrogens >> Rule 5: You should not build waters/alternates at resolutions lower than X >> Rule 6: You should do the final refinement with ALL reflections Rule 7: No >> one cares about getting the carbohydrates right >> >> >> Obviously, this list is not complete. I may also have overstated some of the >> rules to get the discussion going. Any addidtions are welcome. >> >> Cheers, >> Robbie Joosten >> Netherlands Cancer Institute >> >>> Apologies if I have missed a recent relevant thread, but are lists of >>> rules of thumb for model building and refinement? >>> >>> >>> >>> >>> >>> Anthony >>> >>> >>> >>> Anthony Duff Telephone: 02 9717 3493 Mob: 043 189 1076 >>> >>> >>> = > Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoléculas Centro Nacional de Biotecnología - CSIC c/Darwin 3, Campus Cantoblanco 28049 Madrid tel. 91 585 4616 email: mjvanra...@cnb.csic.es
