[ccp4bb] Postdoc position in structural enzymology in Cambridge, UK
Hi all, We have an exciting opportunity for a postdoctoral research scientist to study the physical properties of viral enzymes that allow them to evolve resistance to anti-viral drugs. This project will use advanced computation biology approaches, enzymology and structural biology to define structurally invariant regions of the SARS-CoV-2 MPro protease. This will inform the design of future therapies that reduce/prevent the evolution of drug resistance and may illuminate how evolution of resistance mutations will affect the potency of MPro drugs already in clinical trials. This project is a collaboration between the University of Cambridge (UK) and Rhodes University (South Africa), funded by the Novo Nordisk Foundation as part of the Pandemic Antiviral Discovery (PAD) initiative (see https://padinitiative.com/awarded-grants/rhodes-university-ozlem-tastan-bishop/). The successful candidate will be based in my lab within the Virology Division, Department of Pathology, on the Cambridge Biomedical Campus - Europe's largest biomedical research centre. Cambridge is a great place to work: we have access to shiny instruments for modern biophysical and structural research, we have regular data collection time at Diamond Light Source, there are lots of smart people who you can talk science with, there are old buildings where you can dress up in funny gowns to have dinner in wood-panelled rooms, and it's small enough that you can either walk or cycle to most places you need to be. There's even a river running through it with cows grazing on the banks. If you are a biochemists/enzymologist/structural biologist who wants to work with an international multidisciplinary team to address fundamental research questions with direct relevance to human health and pandemic preparedness, we want to hear from you! Full details of the post and how to apply can be found at https://www.jobs.cam.ac.uk/job/42708/. Please note that you must apply through the website - applications emailed to me can't be considered but I am happy to answer any specific questions you might have. Cheers, Stephen -- Prof. Stephen Graham School of Biological Sciences Theme Leader: Infection and Immunity Department of Pathology University of Cambridge Tennis Court Road Cambridge CB2 1QP http://www.atomicvirology.path.cam.ac.uk/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
Re: [ccp4bb] Bond between Cys S and Met methyl group?
Dear Herman, Great idea - thanks. A quick refinement shows a little bit of positive density between the S atoms, but it certainly isn't screaming out (there's positive density on the other side of the Met S too, both relatively low sigma). So maybe just multiple conformers of the Met and Cys side chains, plus perhaps a close contact of the Met with Cys in one of the conformations. Sadly : ( Thanks all on and off-list for the good ideas! Stephen From: herman.schreu...@sanofi.com Sent: 17 July 2019 15:18 To: Stephen Graham; CCP4BB@JISCMAIL.AC.UK Subject: AW: Bond between Cys S and Met methyl group? Hi Stephen, What happens if you delete the CE methyl group and run a round of refinement. Do you get a positive blob of difference density in between the two sulfurs? Or looks everything pretty ok? In the first case you may indeed have some unusual phenomenon, in the latter case, the methyl group is probably not linked to the cysteine, but at some disordered, invisible position away from the cysteine. This would be the more usual (and boring) explanation. Best, Herman -Ursprüngliche Nachricht- Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Stephen Graham Gesendet: Mittwoch, 17. Juli 2019 16:03 An: CCP4BB@JISCMAIL.AC.UK Betreff: [EXTERNAL] [ccp4bb] Bond between Cys S and Met methyl group? EXTERNAL : Real sender is owner-ccp...@jiscmail.ac.uk Hi all, We've spotted something very weird in our density that we're struggling to reconcile...we have two sulfur atoms (from Met and Cys residues) that are very close together. The density looks for all the world like the methyl group from the Met should also be bonding to the Cys, although obviously the non-bonded terms are keeping them apart during refinement. The dmin is 1.72 and R/RFree are ~0.18/0.20 so the density should be pretty believable. We've seen the same in two crystals. Interestingly, we don't see much change in density if we process just the first/last quarter of the dataset so it isn't overly sensitive to radiation damage during collection. I posted a short vid on Twitter to illustrate: https://urldefense.proofpoint.com/v2/url?u=https-3A__twitter.com_i_status_1151488524425814016=DwIFAw=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=AGJpHgNJ7OJQ60bcbZONEofZkhRwnQqnDQtlX6xRQVY=10R2PgUMqqcCQ2fADadiNWCxz-zY9dt4V8mJt_oPSFE= Has anyone ever seen something like this? Is a (Cys)CA-CB-SG-CE-SD-CG-CB-CA(Met) bond possible? Thanks, Stephen -- Dr Stephen Graham Sir Henry Dale Fellow and University Lecturer Department of Pathology University of Cambridge Tennis Court Road Cambridge CB2 1QP https://urldefense.proofpoint.com/v2/url?u=http-3A__www.path.cam.ac.uk_research_investigators_graham_=DwIFAw=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=AGJpHgNJ7OJQ60bcbZONEofZkhRwnQqnDQtlX6xRQVY=1Wxlqn5ABBEZSTuhDIUatEkDPjr78DB_M6NHrD6xsyY= To unsubscribe from the CCP4BB list, click the following link: https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_webadmin-3FSUBED1-3DCCP4BB-26A-3D1=DwIFAw=Dbf9zoswcQ-CRvvI7VX5j3HvibIuT3ZiarcKl5qtMPo=HK-CY_tL8CLLA93vdywyu3qI70R4H8oHzZyRHMQu1AQ=AGJpHgNJ7OJQ60bcbZONEofZkhRwnQqnDQtlX6xRQVY=LYaCXjAq5Bn667eSDfKBUcrZNF5vmt9FdUxnOiPJ-ps= To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Bond between Cys S and Met methyl group?
Hi all, We've spotted something very weird in our density that we're struggling to reconcile...we have two sulfur atoms (from Met and Cys residues) that are very close together. The density looks for all the world like the methyl group from the Met should also be bonding to the Cys, although obviously the non-bonded terms are keeping them apart during refinement. The dmin is 1.72 and R/RFree are ~0.18/0.20 so the density should be pretty believable. We've seen the same in two crystals. Interestingly, we don't see much change in density if we process just the first/last quarter of the dataset so it isn't overly sensitive to radiation damage during collection. I posted a short vid on Twitter to illustrate: https://twitter.com/i/status/1151488524425814016 Has anyone ever seen something like this? Is a (Cys)CA-CB-SG-CE-SD-CG-CB-CA(Met) bond possible? Thanks, Stephen -- Dr Stephen Graham Sir Henry Dale Fellow and University Lecturer Department of Pathology University of Cambridge Tennis Court Road Cambridge CB2 1QP http://www.path.cam.ac.uk/research/investigators/graham/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
[ccp4bb] Risk assessment for heavy atom soaking - examples?
Hi all, (This email is aimed primarily at my UK colleagues, but feel free to read on and gloat that you don't have to write safety forms in your lab/country!). I need to sort out written risk assessments for heavy atom soaking of crystals in my lab. I wondered whether anyone would be willing to share the risk assessments they have in their institute/company so that I can seek inspiration and make sure I'm keeping up with best practice. Many thanks, Stephen -- Dr Stephen Graham Sir Henry Dale Fellow and University Lecturer Department of Pathology University of Cambridge Tennis Court Road Cambridge CB2 1QP http://www.path.cam.ac.uk/research/investigators/graham/
[ccp4bb] Postdoc in Cambridge on protein:protein interactions in eukaryotic membrane trafficking
Hi all, We’re currently looking for a postdoc to join our lab (http://groups.ds.cam.ac.uk/struvir/) and help us figure out how cells regulate the final steps of endocytosis (i.e. how cells make sure only the right things get sent to lysosomes, the recycling centres of the cell). We’re looking for someone with prior experience in unbiased screening for protein:protein interactions and/or characterisation of such complexes using cell-based, biochemical or structrural/biophysical approaches. Prior experience using proximity-dependent biotinylation (BioID) or studying eukaryotic membrane trafficking would be a plus. Perhaps that's you, or perhaps you have a biochemist/cell-biologist friend who’s heard so many good things about biophysics and protein crystallography from you that they want to give it a shot? More details can be found at http://www.jobs.cam.ac.uk/job/6277/ and applications close on April 27th (only via the website please, it makes the HR people feel unloved otherwise). Cheers, Stephen Dr Stephen Graham Sir Henry Dale Fellow and University Lecturer Department of Pathology University of Cambridge Tennis Court Road Cambridge CB2 1QP Ph: 01223 336920 http://www.path.cam.ac.uk/research/investigators/graham/
[ccp4bb] Postdoc in Cambridge on Virus:Host interactions
Hi there, I have a postdoc job going in my lab to investigate how vaccinia virus (the smallpox vaccine) subverts the innate immune system of infected cells. This project is part of an ongoing collaboration with the (nearby) laboratory of Prof. Geoff Smith and the successful candidate will get to work closely with the virologists in Geoff’s lab in order to characterise these proteins: from solving atomic-resolution structures right through to probing their role in virulence of the virus. Examples of the kind of things we’re looking at are (DOIs): 10.1371/journal.ppat.1003183, 10.7554/eLife.00047 and 10.1074/jbc.M114.624650 More details of the post and how to apply can be found here: http://www.jobs.cam.ac.uk/job/5852/ Please note that (by HR diktat) I’m only allowed to consider applications submitted via the online application form - you’ll need to note in your cover letter that you’re applying for Position 3. Applications close on Tuesday, so there’s no time to waste! Cheers, Stephen Dr Stephen Graham Sir Henry Dale Fellow and University Lecturer Department of Pathology University of Cambridge Tennis Court Road Cambridge CB2 1QP Ph: 01223 336920 http://www.path.cam.ac.uk/research/investigators/graham/
Re: [ccp4bb] Off Topic: Isothermal Titration Calorimetry (ITC)
We bought a NanoITC from TA instruments last year and I can't recommend it enough. We are able to get excellent binding curves with the low volume cell/syringe (200uL/50uL, respectively, which actually means 350uL/120uL in order to load both without bubbles) using the same concentrations of protein as we used to use in a MicroCal VP-ITC. The control/analysis software is much easier to use (although the graphing capabilities could be improved) and the technical support has thus far has been excellent. Also, as others have said it's a steal compared with the equivalent GE machine (both in terms of purchase cost and cost of maintenance contracts). Stephen On 17 January 2012 16:02, Jose Artur Brito jbr...@itqb.unl.pt wrote: Dear All, sorry for this off-topic questions but I would like to have some feed-back from you on Isothermal Titration Calorimetry (ITC) equipments. We have a very nice quotation for an iTC200 from GE Healthcare. We wanted this one because it uses ~200uL sample per measurement (really nice when your dealing with precious samples, ie., proteins with low expression yields). However, I was told that, although consuming much less sample, is not as good (sensitivity, mixing issues, bubbles, ...), as the VP-ITC (it uses ~1.4mL per measurement, seven times more than the iTC200). Does anyone has experience with these two equipments? Would you prefer one over the other (please state your reasons)? Would you suggest another equipment/brand for the ITC (like the NanoITC from TA Instruments)? Thanks in advance, Jose Artur Brito -- * José Artur Brito, PhD * * * * Post-Doctoral Fellow * * Membrane Protein Crystallography Lab * * Instituto de Tecnologia Química e Biológica * * Oeiras - Portugal * * * * Tel.: +351.21.446.97.61 * * Fax: +351.21.443.36.44 * * * * E-mail: jbr...@itqb.unl.pt * * URL: http://mx.itqb.unl.pt * -- Dr Stephen Graham 1851 Research Fellow Cambridge Institute for Medical Research Wellcome Trust/MRC Building Addenbrooke's Hospital, Hills Road Cambridge, CB2 0XY, UK Phone: +44 1223 762 638
Re: [ccp4bb] non-waters among structured solvent atoms
You might also want to try: http://www.ncbi.nlm.nih.gov/pubmed?term=12499536 Cheers, Stephen On 15 June 2011 02:09, Robbie Joosten robbie_joos...@hotmail.com wrote: Hi Wolfram, This was an early study on the subject: http://www.ncbi.nlm.nih.gov/pubmed/8594192 The software is still accessible via the STAN server. Cheers, Robbie Date: Tue, 14 Jun 2011 17:51:21 -0400 From: wtem...@gmail.com Subject: [ccp4bb] non-waters among structured solvent atoms To: CCP4BB@JISCMAIL.AC.UK Dear colleagues, following a discussion in our lab, I have volunteered to dig out articles from the literature about erroneous assignments of non-water entities such as metal ions, halides in protein models. For example I have the faint recollection that data mining of the PDB for suspect water assemblies matching the geometry of coordinated cations has previously been described. But none of my google searches has turned up the references I was looking for. Could someone point me in the right direction, please? Many thanks, Wolfram Tempel -- Dr Stephen Graham 1851 Research Fellow Cambridge Institute for Medical Research Wellcome Trust/MRC Building Addenbrooke's Hospital, Hills Road Cambridge, CB2 0XY, UK Phone: +44 1223 762 638
Re: [ccp4bb] Per-residue RMSD for multiple structures?
I am pretty sure you can do this using LSQMAN from Gerard (BluRay?) Kleywegt. The pertinent commands are MCENTRAL to determine the 'most representative structure' (i.e. the one to align upon and show in the figure), MALIGN to do the alignment and then MPLOT to calculate a 'multi-RMSD' for each residue (see manual for details - set the 'cut-off for printing' to 0 to get all values). Regards depiction, I think pymol can also represent structures as sausages based on their B values: cartoon putty show cartoon HTH, Stephen On 23 February 2010 01:31, Ethan Merritt merr...@u.washington.edu wrote: Hi all, I am comparing 4 very similar (1.5A rmsd) large (750 residues) structures, but struggling to find a way to generate a figure that conveys where they are most alike and where they diverge. Simply drawing a superimposed set of backbone traces results in what looks like colored spaghetti. I don't think that's going to work. So I had the idea of drawing a single backbone trace, or ribbon diagram, and coloring by the RMSD of the four C-alphas at each residue position. But I can't find a program that will output this as a table of numbers I can use. All of the multiple structure superposition programs must have this information internally. After all, that's what they are minimizing. But do any of the programs provide an option to write it out? I can get pairwise per-residue deviations by doing SSM superposition in Coot, but that doesn't get me to an RMSD for all four structures jointly. Ethan -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742 -- Dr Stephen Graham 1851 Research Fellow Cambridge Institute for Medical Research Wellcome Trust/MRC Building Addenbrooke's Hospital, Hills Road Cambridge, CB2 0XY, UK Phone: +44 1223 762 638
Re: [ccp4bb] Anions in protein structures
A good place to start is P. Müller, S. Köpke and G. M. Sheldrick (2003) Is the bond-valence method able to identify metal atoms in protein structures? Acta Cryst. (2003). D59, 32-37[ doi:10.1107/S0907444902018000 ] Cheers, Stephen 2010/1/5 Clayton, Gina Martyn gina_clay...@merck.com: Dear CCP4ers Can anyone recommend their favourite paper, or review, that discusses anions in protein structures. Thanks in advance and Happy New Year Gina 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. -- Dr Stephen Graham 1851 Research Fellow Cambridge Institute for Medical Research Wellcome Trust/MRC Building Addenbrooke's Hospital, Hills Road Cambridge, CB2 0XY, UK Phone: +44 1223 762 638
Re: [ccp4bb] Crystallization of lysine and arginine rich proteins
Try reductive methylation of the lysines: Walter, T. S., Meier, C., Assenberg, R., Au, K. F., Ren, J., Verma, A., Nettleship, J. E., Owens, R. J., Stuart, D. I. Grimes, J. M. (2006). Structure, 14, 1617–1622. Cheers, Stephen 2009/11/2 Jan Rash jan...@googlemail.com: Dear All, I have a question regarding the crystallization of lysine and arginine rich protein around 13%. So far our attempts to crystallize this protein have not been successful although the secondary structure predictions, CD spectroscopy measurements clearly show that this protein is folded. I presume that these lysine and arginine are the sources of the local flexibility in the protein even though the protein is globular overall. Moreover, my attempts to crystallize the limited proteolysis fragments also did not achieve crystals. I have also tried the crystallization with its binding partners and could not succeed. I think any compound that binds to the lysine/arginine side chains might affect the crystallization process thereby reducing the internal flexibility of protein. Can anybody suggest some effective strategy for the crystallization? Thanks Umar -- Dr Stephen Graham 1851 Research Fellow Cambridge Institute for Medical Research Wellcome Trust/MRC Building Addenbrooke's Hospital, Hills Road Cambridge, CB2 0XY, UK Phone: +44 1223 762 638
Re: [ccp4bb] off topic; alignment keeping upper and lower case
Charlie Bond's ALINE program (http://crystal.bcs.uwa.edu.au/px/charlie/software/aline/) can handle upper/lower case sequences. In the menu bar: Tools Alignment Align All Sequences does the job (using clustalw as a back-end IIRC) without changing cases. Best, Stephen 2009/9/16 MARTYN SYMMONS martainn_oshioma...@btinternet.com: Dear All - I am looking for a multiple alignment program that will work with upper and lower case letters in the sequences and preserve them through to the output. I used to use PILEUP but that is no longer supported here. Any suggestions much appreciated. regards Martyn Martyn F. Symmons Department of Pathology University of Cambridge 'Gun fhaillin 'san fhairge..' (said of Griomasaigh boats) -- Dr Stephen Graham Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] European IPTG suppliers
We get most of our 'standard' lab chemicals (IPTG, Tris, etc.) from Melford - http://www.melford.co.uk For IPTG: 1 gm8.00 ₤ 5 gm25.00 ₤ 10 gm 45.00 ₤ 25 gm 105.00 ₤ 50 gm 195.00 ₤ 100 gm 360.00 ₤ Cheers, Stephen 2009/7/13 Mark J. van Raaij mark.vanra...@usc.es: Dear All, as a structural biology lab, we use quite a bit of IPTG...not cheap and needs to be of sufficient quality for bacterial protein expression induction. Therefore I thought it would be useful to compare supplier prices in Euros, realising that of course pricing may vary across different countries and in time. Personally, I am only interesting in EU (Schengen) suppliers, because ordering from outside is too complicated for us. But I will make a compilation of replies and include other ones. Comments about negociable large quantity orders (up to 100 g) are also welcome. Greetings, Mark IPTG prices in Spain, approx. without tax. Qiagen (via Izasa): 5 g 142 euros Promega: 50 g 1140 euros, 5 g 181 euros, 1 g 61 euros Sigma: 10 g 494 euros, 5 g 250 euros Invitrogen: 1 g, 44 euros Mark J. van Raaij Dpto de Bioquímica, Facultad de Farmacia Universidad de Santiago 15782 Santiago de Compostela Spain http://web.usc.es/~vanraaij/ -- Dr Stephen Graham Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] My cysteines
How about an oxidised cysteine? Sulfenic acid is a possibility (http://en.wikipedia.org/wiki/Sulfenic_acid), although it will generally oxidise further to sulfonic acid (http://en.wikipedia.org/wiki/Sulfonic_acid). I've seen them before in structures of old (4-5 year old) crystals (see figure 2 of Biochemistry, 2005, 44 (42), pp 13820–13836. DOI: 10.1021/bi0512849). Cheers, Stephen 2009/4/21 James Stroud xtald...@gmail.com: Hello All, I have a couple of cysteines with some extra density about 1 covalent bond's length away from the sulfur center. It looks to be one atom's worth of extra density. Because I could fit it in an icon sized graphic and I anticipate that someone will suggest I post a picture, I'm attaching a picture of the positive fofc density. Does anyone have any idea of the usual culprits here? I see no negative density in the region. James -- Dr Stephen Graham Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] difficult MAD dataset
Chi2 is unusually high at lower resolution (Chi2 is 3 from 3.5A as shown below) and there is a relatively high percentage of rejections (1.5 %). Chi^2 in Scalepack *must* be adjusted by changing the expected errors to be about 1. Until then, I would not reject reflections. Only then I would reject reflections and then readjust chi^2 to be 1 again. Having said that its quite a while since I used scalepack. Maybe in the age of automation scalepack does this now automagically in the context of HKL2000, but a Chi^2 of 3 at low resolution indicates that some reflections and very likely their sigmas do not have realistic intensities, and also that your rejections might be excessive. Just a point here. The high chi^2 values you see in the scalepack output are because of the *anomalous signal*, not because of any pathology with the data. I assume scalepack was run with the 'anomalous' card specified. In this case (I believe) the + and - reflections will be treated as equivalent for scaling and calculating statistics, but will be written to the .sca file separately. Chi^2 will be screwed up because it detects a large deviation from random in the reflections (i.e. the anomalous difference between I(+) and I(-). You might actually be rejecting the reflections with the *most* anomalous signal since they are the ones that deviate furthest from your random-noise model. If you want your chi^2 values to be meaningful you need to use the 'scale anomalous' card to separate I(+) and I(-) for scaling/statistics as well as the output .sca. The scalepack manual warns against this flag, but I've never had a problem using it (so long as you have reasonably redundant data). So, re-process with 'scale anomalous' and then run autoSHARP in all sub-spacegroups of P212121 - as Tassos will tell you this works a treat! Cheers, Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] Google marks CCP4 web site as a potential security threat
Google gives phenix-online.org, cns.csb.yale.edu and globalphasing.com the same treatment, so at least they can't be accused of bias towards a particular software suite. Or even a technique, as the ARIA and GROMACS websites are similarly tarred. A plot by Google against Structural Biology in general? Stephen On Jan 31, 2009, at 9:50 AM, Miguel Ortiz Lombardia wrote: Dear all, While searching for some definition of rotations angles I have bumped into this very disagreeable indeed discovery: google adds now a step if you want to go to places _they_ consider as potentially dangerous. You can proceed that have to copy and paste the address yourself or tell the relevant webmasters to do whatever Google wants them to do so their pages don't appear as risky. One such place happens to be the full ccp4 web site. In my opinion, the real danger is Google. Time to switch to another search engine? In any case, I wanted CCP4 developers and users to know. ( the search was done from a home adsl connection in France ) Best regards, Miguel -- Miguel Ortiz Lombardía Architecture et Fonction des Macromolécules Biologiques UMR6098 ( CNRS, U. de Provence, U. de la Méditerranée ) Case 932 163 Avenue de Luminy 13288 Marseille cedex 9 France Tel : +33(0) 491 82 55 93 Fax: +33(0) 491 26 67 20 e-mail: miguel.ortiz-lombar...@afmb.univ-mrs.fr Web: http://www.pangea.org/mol/spip.php?rubrique2 -- This message has been scanned for viruses and dangerous content by MailScanner, and is believed to be clean. -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] problem of crystallization
Try reductive methylation of surface lysines. http://dx.doi.org/10.1016/j.str.2006.09.005 Cheers, Stephen On 5/13/08, Jennifer Han-Chun Tsai [EMAIL PROTECTED] wrote: Hi, This topic is not related to CCP4. I am having problem of crystallizing one protein. It's a pretty small protein with size around 15kDa. I have stock concentration around 100mg/mL. Crystallization plates I set up are with concentration of 10mg/mL, 30mg/mL and 50mg/mL. All the plates had been set up at least one week. Only around 5 wells per plate or less formed precipitation. The rest of wells are pretty clear still. Is there any suggestion for reducing protein solubility or increasing the chance of getting crystals? Thanks for your time, Jennifer -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] discontinuous data wedges in XDS
Hi Bert, xia2 is your friend in cases like this. This program is a real boon for the lazy crystallographer. All you need to type is: xia2 -3d /path/to/images and xia2 will automagically index, integrate and scale all of the sweeps together. Add the -atom Se flag (atom name as appropriate) to be sure you keep anomalous signal in the final mtz file. See http://www.ccp4.ac.uk/xia/ for more info. Cheers, Stephen On 4/29/08, Van Den Berg, Bert [EMAIL PROTECTED] wrote: Hi all, is it possible to input discontinuous data wedges into XDS (obtained from for example inverse beam sweeps)? (So wedge se1 goes from 0-90 deg (image 1-90), se2 from 180-270 (image 1-90), etc). Or do I have to rename everything so that I get one data file in which the rotation ranges are continuous? Thanks, Bert Bert van den Berg University of Massachusetts Medical School Program in Molecular Medicine Biotech II, 373 Plantation Street, Suite 115 Worcester MA 01605 Phone: 508 856 1201 (office); 508 856 1211 (lab) e-mail: [EMAIL PROTECTED] http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
[ccp4bb] multiple sequence alignment from multiple pairwise structural alignments
Hi all, I would like to generate a structure-based multiple sequence alignment using 4 structures. I have already generated pairwise alignments for each 'pair' of structures (6 alignments in all). Is there a program out there that can take a number of aligned structures (or even just a number of pairwise sequence alignments) and calculate the 'best' multiple sequence alignment? Please note that there is absolutely no sequence conservation between these structures, making standard sequence-based alignment tools pretty useless. Thanks, Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] anomalous signal of Mn and Ca ions
As Ethan mentions, your best bet for an 100% positive ID would be to collect a dataset above and below the Mn edge and see the anomalous signal from the Mn atom disappear. If that is not an option, there is a great paper by George Sheldrick and friends regards using the Calcium Bond Valence Sum to determine the nature of a metal based on its coordination geometry. [P. Müller, S. Köpke and G. M. Sheldrick (2003) Is the bond-valence method able to identify metal atoms in protein structures? Acta Cryst. D59, 32-37]. shameless self-plug You could also have a look at one of my papers where we applied this (and integration of the anomalous difference density around the position of the scatterer, scaled against the Sulfur signal of the Met's and Cys'es in the protein) to confirm metal loading of a metalloenzyme [Graham, S, Bond, C, Freeman, H, Guss, J. (2005) Structural and functional implications of metal ion selection in aminopeptidase P, a metalloprotease with a dinuclear metal center. Biochemistry.44: 13820-36] Good luck, Stephen On 2/29/08, Sun Tang [EMAIL PROTECTED] wrote: Dear All, In my structures, I want to assign Mn or Ca ions for some densities. But when I did not have anomalous density in CCP4i. I am not sure whether I was correct. The following was what I did: I processed the data with HKL2000 and select anomalous signal in scaling. In CCP4i, I selected Run FFT-Creat Map in the Map Mask Utilities. I select O format to cover asymmetric unit and Plot section on Z axis from 0 to 1 in steps on 10. All others were by default values. I display in ono10. I collected the data at the wavelength of 1 A. Do I need to adjust the wavelength to maximize the anomalous signal from Mn or Ca? Any ideas and suggestions are greatly appreciated! Sun Tang Never miss a thing. Make Yahoo your homepage. -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] Vary b-factors from a plane/point.
Hi David, Attached is a script I wrote for doing a similar thing in pymol. It sets the B value of an atom to be the distance of that atom from a specified point (optionally normalising the final values from 0-100). You can then either draw the molecule coloured by B in pymol, or export the coords for use in your favourite graphics package. Usage is as follows (from inside pymol, assuming the script is in directory /path/to): run /path/to/distance_to_B.py distance_to_B( all, [0,0,0] ) It would be fairly trivial to extend the code to measure the distance from a plane instead of from a point. Cheers, Stephen On 10/9/07, David Briggs [EMAIL PROTECTED] wrote: Hi everyone, I know that this has been discussed on the bb before now, but 20 pages of google search in, I have still come up with zip. I want to vary b-factors smoothly from either point or a plane. This is for pretty-picture purposes. I can then colour molecule by b-factor and get nice, pretty effects. Thank you in advance for your assistance, David -- David C. Briggs PhD Father Crystallographer http://personalpages.manchester.ac.uk/staff/David.C.Briggs/ BROKEN! AIM ID: dbassophile -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549 from pymol import cmd from pymol import stored from math import * def distance_to_B( selection, origin, state=1, normalise=1 ): stored.xyz = [] cmd.iterate_state( state, selection, stored.xyz.append([x,y,z]) ) x0, y0, z0 = origin[0], origin[1], origin[2] distances = [] for i in stored.xyz: x = i[0] y = i[1] z = i[2] distances.append( sqrt((x-x0)**2+(y-y0)**2+(z-z0)**2) ) sorted_dists = distances[:] sorted_dists.sort() min_dist = sorted_dists.pop(0) max_dist = sorted_dists.pop() range = max_dist - min_dist stored.b = [] for i in distances: if normalise == 1: stored.b.append( ((i - min_dist)/range)*100 ) else: stored.b.append( i ) cmd.alter( selection, b=stored.b.pop(0) ) cmd.extend( distance_to_B, distance_to_B )
Re: [ccp4bb] extra density on Cysteine
2cystein.jpeg looks just like oxidation of cysteine to S-hydroxy-cysteine (a.k.a. cysteine sulfenic acid). I have seen this repeatedly in one of my structures (E. coli aminopeptidase P, see 1WL9 for an example). We discuss this a bit in Graham et al (2005) Biochemistry 44: 13820-36 - see Figure 2 and surrounding text. Cheers, Stephen On 8/14/07, [EMAIL PROTECTED] [EMAIL PROTECTED] wrote: Dear all, I am refining a 2.0A structure. I found that there were some extra density on two cysteines, even though I have added 5mM BME in the protein buffer. I am wondering whether the first one (Cys292) is a bme and the second one is an oxidized cysteine. Any suggestion? I attached the images for your reference. thanks Regards _ Xu Ting ,Ph.D 10 Biopolis Road Singapore 138670 Fax: +65 6722 2916 Phone: +65 6722 2980 -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] highly soluble proteins
Reductive methylation of amino groups (lysines and the N-terminus) is a fairly routine chemical modification that should drop the solubility of your protein. An easy-to-use protocol can be found in Walter et al (2007) Lysine Methylation as a Routine Rescue Strategy for Protein Crystallization. Structure, 14:1617-1622 Cheers, Stephen On 8/7/07, [EMAIL PROTECTED] [EMAIL PROTECTED] wrote: Yvonne, Several 'old' proteins have been crystallized from insanely high concentraitons - concanavalin A for instance can be grown from 100-250 mg/ml solutions by means of 'salting in' using microdialysis. This is of course highly labor-intensive and also expensive on the protein side. Look at the distribution of charged residues on the protein (especially helpful if you have a model of some sort). I would recommend mutating some of them to non-charged or even nonpolar residues in order to lower solubility. Alternatively, you could try preparing chemical derivatives of the protein using e.g. heavy atoms or amine-modifying reagents like NHS. If you block enough amines, your solubility should go down and your pI will change as well. Likewise you could try modifying exposed acidic residues etc. Mutagenesis can in the end be easier to do because chemical modification tends to produce complex mixtures of products, unless you do it with a huge excess of the reagent and allow the reaction to proceed to exhaustion. Artem Our lab is trying to crystallize a highly soluble (100+ mg/ml) protein with a molecular weight of 35 kd. The protein was screened against 1536 conditions at 20 mg/mL. Most drops were either clear or produced bubbles (often oily looking). The few that had precipitate contained high concentrations of K3PO4, cobalt, or zinc. We have tried repeating some of the bubble conditions at 100+ mg/mL and are still getting clear drops or bubbles. Is there something about highly soluble proteins and/or secreted proteins and/or proteins with unusual portions of their sequence that needs to be considered in order to successfully crystallize it? I am considering trying salting out using dialysis, and also adding ligands/inhibitors. The protein is in 50 mM NaCl plus 50 mM buffer. I welcome thoughts and suggestions on crystallization ideas, publications, etc. Thank you Yvonne -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] Stereo grpahics and Virtual Reality
Full VR systems with motion tracking and are now affordable to most labs (~$2500 for all the peripherals). Nintendo Wii remotes are about £25 and they do 3-dimensional motion tracking. Perhaps we should by trying to use them for refinement instead? They communicate via the Bluetooth protocol (http://www.wiili.org/Wiimote) and have already been used for controlling industrial robots (http://www.youtube.com/watch?v=0qEotHQgUsg). Stephen -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
Re: [ccp4bb] Differentiating bound Mn Ca.
Hi David, You can use Sheldrick's Calcium Bond Valence Sum to descriminate between metals (see Muller, P., Kopke, S., and Sheldrick, G. M. (2003) Acta Crystallogr., Sect. D: Biol. Crystallogr. 59, 32-37) even at low resolution. I have had good success with this method combined with estimation of anomalous contribution scaled against the S atoms as suggested by Kay. Have a look at gratuitous self plug Graham, S. C., Bond, C. S., Freeman, H. C., and Guss, J. M. (2005) Biochemistry 44, 13820-13836. /gratuitous self plug for plenty of examples. To do the anomalous difference calculations I just integrated a 2x2x2 A box around the metal atom and around the S atoms (in MAPMAN) then calculated the ratio of anomalous difference... Cheers, Stephen On 4/16/07, David Briggs [EMAIL PROTECTED] wrote: Dear all. I have recently solved a structure in-house, 2.8A, CuKa. I have a metal ion bound very obvious hepta-valent co-ordination, which would suggest either Ca or Mn. Neither was present in the crystallisation setup, but there was some Mg around, which has contaminants of both Ca Mn. At 2.8A, I don't really think I can reliably discriminate between 2.15A 2.36A distances to coordinating atoms (http://tanna.bch.ed.ac.uk/newtargs_06.html ). The B factors for refined Ca are 18, and Mn 30. The B-factors of coordinating atoms vary from... 18 30 - so no help there. I have a nice clear 6sigma anomalous difference peak, but then, according to http://skuld.bmsc.washington.edu/scatter/ both Ca (f ~1.3) and Mn (f ~2.8) scatter anomalously at that wavelength. The obvious solution is go to a synchrotron and scan around the Mn edge and see what happens, however, whilst waiting for beam time, is there any way I could... oh I don't know, use the peak in my anomalous difference Fourier to figure out what anomalous signal would be required to generate a peak of that size - a sort of back-transform??? Is this do-able, and if so, how would one go about it? Cheers, Dave -- --- David Briggs, PhD. Father Crystallographer www.dbriggs.talktalk.net iChat AIM ID: DBassophile --- Anyone who is capable of getting themselves made President should on no account be allowed to do the job. - Douglas Adams -- Dr Stephen Graham Nuffield Medical Fellow Division of Structural Biology Wellcome Trust Centre for Human Genetics Roosevelt Drive Oxford OX3 7BN United Kingdom Phone: +44 1865 287 549
