Re: [ccp4bb] Crick-Magdoff and anomalous
Ethan Merritt wrote: Please also have a look at A Olczak, M Cianci, Q Hao, PJ Rizkallah, J Raferty, JR Helliwell (2003). S-SWAT (softer single-wavelength anomalous technique) Acta Cryst. A59, 327-334. in which the authors show several derivations for the estimated anomalous signal, based on slightly different assumptions. And the generalization of their formulae is given in : Flack, H. D. Shmueli, U. (2007). Acta Cryst. A63, 257-265. In a follow-up paper, their derivations were extended to all spacegroups, also taking account of special reflections. -- Marc SCHILTZ http://lcr.epfl.ch
Re: [ccp4bb] refmac not rerunnable
As far as I know the compatibility has been broken because of adding new features such as intensity based refinement or multiple anomalous scatterers to the GUI Pavol -- Sent from: Leiden ZH Netherlands. This is VERY VERY VERY irritating! Why has it been allowed... Is there any advantages?? Eleanor#
Re: [ccp4bb] Problems with phasing a protein (1300aa)
Hi - I must agree that if one RTFMs its clear. Now, back to the real world. My experience is that most SCALA users tend to look at I/sigmaI. I admit that I had been using that for deciding data cutoff, many times, but thats another discussion. The vast majority of Table 1 I see report I/sigmaI, with never clarifying if thats I/sigma(I) or I/sigma(I). Anyway ... SCALA indeed clearly says SIGMA :- rms scatter of observations sd :- average standard deviation derived from experimental SDs, after XDS clearly says as well: I/SIGMA = mean of intensity/Sigma(I) of unique reflections (after merging symmetry-related observations) so, if I understand it right, SCALA and XDS use for a different metric the same shorthand label, even if they indeed clarify what it is in each case. A user would tend to look in both cases to I/sigma (since thats the jargon that prevailed) d*TREK could confuse people further as to which one should be used for reporting and decision making. Scalepack conveniently ignores the problem by not reporting the I/ sigma, but still lets you wonder if you should divide the I with the average error or the Average 'stat.'. (I am still confused btw of what one should do). The Big Question Again: Which the infamous I/sigma that should be above 2.0 and should be in Table 1 ...? If we agree we want I/sigma(I) (after sigmas are corrected I presume) here is a solution: Since Phil is generous enough to offer to rename the Mn(I/sd) column to I/SIGMA and maybe rename I/sigma to I/rms-scatter or so, if Jim could also rename one of his columns, and Wladek can add one, we can have a standard. Or even better if everybody would use I/sigma(I) then we would even start getting the tables right in the papers? (... I could see this train of thought going out of its rails while I was typing ...) Tassos On Mar 23, 2009, at 17:08, Phil Evans wrote: I'm happy to change the column titles if it makes it clearer. Actually the I/sigma column in the Scala output is not very useful: it is I / RMSscatter, ie the mean intensity/mean error, for individual observations, not taking into account multiple measurements. Because it is ratio of means (rather than a mean of ratios), it can behave oddly depending on the distribution of intensities, for instance giving an overall value which is outside the range of values in resolution bins. It is the ratio of the previous two columns. On the other hand the column labelled Mn(I)/sd is the mean of ratios for each reflection, ie I/σ(I) and does take into account the multiplicity of measurements, so is much more relevant as an indicator of data quality see http://www.ccp4wiki.org/~ccp4wiki/wiki/index.php?title=Scaling_experimental_intensities_with_Scala Scala also outputs a convenient Table 1 summary On 23 Mar 2009, at 15:50, James Holton wrote: I guess when I talk about signal-to-noise I assume the one that is most relevant to the task at hand. So, to me, I/sigma(I) at the phasing step would be the average intensity (I) divided by the sigma (standard deviation) assigned to it AFTER scaling/mergeing. I admit that the I/sigma column from SCALA is potentially confusing, but if you are dealing with spot intensities, this is the first I/sigma you think about, so I guess this is what Phil was thinking. Personally, I think the descriptions of the columns in this table are clear if you read the caption before the table in the SCALA output, but Tassos is right that an alarming number of people have never done this. RTFM? When it doubt, use mtzdmp to see what the average values of the data columns really are. -James Holton MAD Scientist Anastassis Perrakis wrote: I like to think of things in terms of signal-to-noise, and one can use a rearrangement of the Crick-Magdoff equation to tell you what the I/ sigma of your data set needs to be for delta-F to be greater than sigma(delta-F): I/sigma(I) 1.3*sqrt(Daltons/sites)/f where: I/sigma(I) is the signal-to-noise ratio of the data set required to solve it by MAD/SAD Daltons is the molecular weight of the protein in amu sites is the number of Se sites fis the f of those sites (in electrons) let me see we recently solved a 200 residues protein, 4 mol AU, with 2 Se per mol, total 8 Se. Since 160 residues were ordered, I will make for you a discount, 18,000 D/monomer, 70,000 in AU. I truncated data to 4.2 for Se search. 1.3*sqrt(7/8)/6.5= 19 Statistics from Scala: N 1/d^2 Dmin(A) Rmrg Rfull Rcum Ranom NanomAv_I SIGMA I/ sigma sd Mn(I/sd)1 0.0098 10.11 0.048 0.049 0.048 0.036349 4967419 11.9342 25.8 2 0.0196 7.15 0.050 0.044 0.049 0.031707 5360462 11.6 372 28.8 3 0.0293 5.84 0.089 0.062 0.057 0.047 975 1634224 7.3177 19.4 4 0.0391 5.06 0.065 0.048 0.059 0.039 1140 2107207 10.2218 21.0 5 0.0489
[ccp4bb] misdirected email
Dear all, I am terribly sorry about my mis-directed email. Please accept my sincere apologies. Manfred. -- * * *Dr. Manfred S. Weiss * * * * Team Leader * * * * EMBL Hamburg OutstationFon: +49-40-89902-170 * * c/o DESY, Notkestr. 85 Fax: +49-40-89902-149 * * D-22603 Hamburg Email: mswe...@embl-hamburg.de * * GERMANY Web: www.embl-hamburg.de/~msweiss/ * * *
[ccp4bb] postdoctoral position available in Cambridge
Postdoctoral Vacancy Research Associate A Wellcome Trust funded postdoctoral position is available to work with Dr Matt Higgins in the Department of Biochemistry, Cambridge University on the structural biology and biochemistry of cell surface proteins involved in severe and cerebral malaria. Experience in protein expression, purification and crystallography would be a major asset for this position. Applications are welcomed from talented scientists, who hold (or will be shortly be receiving) a PhD in a relevant biological discipline. Applications should include a covering letter describing relevant research experience to date, a CV, PD18 form with parts 1 and 3 completed and the names and addresses of two referees. These should be sent quoting the reference by email by to j...@bioc.cam.ac.uk or post to The Principal Assistant, Department of Biochemistry, Building O, Downing Site, Tennis Court Road, Cambridge, UK, CB2 1QW. Informal enquiries can be made to Matt Higgins on mk...@cam.ac.uk Salary: 27,183-35,469 pa Limit of Tenure 36 months in the first instance Quote Reference: PH04897 Closing Date: 3 April 2009
Re: [ccp4bb] Problems with phasing a protein (1300aa)
Hi Tassos, It's a bit early in the year to have our annual I/sigI discussion on the CCP4BB, isn't it? Usually, we wait until at least April 1. :-) . d*TREK could confuse people further as to which one should be used for reporting and decision making. . The Big Question Again: Which the infamous I/sigma that should be above 2.0 and should be in Table 1 ...? Since d*TREK gives you a Table 1 to place in the supplementary material of your paper where no one can read it, you should use that one. If we agree we want I/sigma(I) (after sigmas are corrected I presume) here is a solution: Since Phil is generous enough to offer to rename the Mn(I/sd) column to I/SIGMA and maybe rename I/sigma to I/rms-scatter or so, if Jim could also rename one of his columns, and Wladek can add one, we can have a standard. Or even better if everybody would use I/sigma(I) then we would even start getting the tables right in the papers? I got no problem changing labels. In d*TREK dtscaleaverage one column is the mean I/sigma(I) of the input observations, one column is the mean I/sigma(I) of the output unique reflections. This is clearly stated in the output and one does not have to read the manual. I believe these are identical to SCALA's columns and do convey useful information. Jim
[ccp4bb] two identical proteins in one asymmetric unit
I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710
[ccp4bb] Practical Workshop on Characterization of Protein Complexes in Structural Biology
Dear all, I would like to draw your attention to the following scientific event in preparation: Practical Workshop on Characterization of Protein Complexes in Structural Biology EMBL-Hamburg, 30th June - 3rd July 2009. The workshop is aimed at advanced graduate students and postdoctoral fellows interested in expanding their knowledge of state-of-the-art biophysical and biochemical characterization methods of protein complexes. The workshop will include hands-on sessions and lectures by world-leading experts from relevant fields. Topics covered will include FRET, BIFC, SAXS, ITC, high-throughput crystallization and mass spectrometry. The course is supported by the EU funded projects SPINE2-complexes (http://www.spine2.eu/SPINE2/), 3D Repertoire (http://www.3drepertoire.org/) and PENELOPE (http://penelope.crg.es/). The course is open to everyone, however several places have been reserved for members of the supporting grants. There is no registration fee and accommodation and subsistence are covered for accepted applicants. Accommodation will be provided on the basis of shared double rooms. The deadline for applications is the 30th of April 2009. A letter of reference will be required from applicants not associated with a supporting grant. A selection committee will consider the applications and the result of the selection procedure will be sent to applicants by Email within about three weeks of the closing date. Please note that it is the responsibility of the applicant to ensure that a letter of reference is uploaded prior to the application deadline. For more information please visit the webpage: http://www.embl-hamburg.de/protchar2009 On behalf of the organizing committee, Vivian POGENBERG EMBL-HAMBURG
Re: [ccp4bb] Problems with phasing a protein (1300aa)
Since Phil is generous enough to offer to rename the Mn(I/sd) column to I/SIGMA and maybe rename I/sigma to I/rms-scatter or so, if Jim could also rename one of his columns, and Wladek can add one, we can have a standard. Or even better if everybody would use I/sigma(I) then we would even start getting the tables right in the papers? I got no problem changing labels. In d*TREK dtscaleaverage one column is the mean I/sigma(I) of the input observations, one column is the mean I/sigma(I) of the output unique reflections. This is clearly stated in the output and one does not have to read the manual. I believe these are identical to SCALA’s columns and do convey useful information. I clearly agree, that all that info convey useful information. SCALA as well as clearly document these as well. My point was mostly that XDS and SCALA use the same 'shorthand' to report different things, which is confusing. I then thought that if Phil changes labels as he was willing, it could be good if all of you guys would agree in one label for the same thing... Up to you of course! I will now start thinking of a good April 1 email ... Best, Tassos
Re: [ccp4bb] two identical proteins in one asymmetric unit
Dear Sang They are really different! And I guess you would probably want to use NCS restraints depending on your resolution. Regards, Folmer 2009/3/24 Sang Hoon Joo s...@duke.edu: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710
[ccp4bb] OT: Crystallisation compatible detergents
Hello, We can make a nice 1:1 complex between two proteins that then gradually precipitates during storage at 4 degC. With 0.005% Tween-20 the complex is stable and does not preciptate noticeably. We have used this in buffers to measure the kinetics of binding by Biacore so it is clearly compatible with functionality. Is Tween-20 at this concentration compatible with crystallisation? Is it worth giving a go, or a waste of time? Should we try and remove it, or reprepare the complex in an alternative detergent? If we need to try an alternative, what would people recommend? Thanks in advance for your suggestions, Darren -- ** Dr. Darren Hart, Team Leader High Throughput Protein Lab Grenoble Outstation European Molecular Biology Laboratory (EMBL) ** EMBL webpages: http://tinyurl.com/6xdltl http://tinyurl.com/667jrp Email: h...@embl.fr Tel: +33 4 76 20 77 68 Fax: +33 4 76 20 71 99 Skype: hartdarren Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex 9, France **
Re: [ccp4bb] two identical proteins in one asymmetric unit
Sang Hoon, Each molecule in the asymmetric unit is most likely different. I work on a protein that crystallizes as a homodimer with 2 molecules per asymmetric unit and there are some differences between the two (eg: electron density visible for the 14 N-terminal residues in one molecule, but not the other). Cheers, Jim On Tue, Mar 24, 2009 at 11:03 AM, Folmer Fredslund folm...@gmail.comwrote: Dear Sang They are really different! And I guess you would probably want to use NCS restraints depending on your resolution. Regards, Folmer 2009/3/24 Sang Hoon Joo s...@duke.edu: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710 -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
Re: [ccp4bb] two identical proteins in one asymmetric unit
Dear SHJ, there is no reason to expect that the 3D structure of two molecules within the same asymmetric unit are identical even if their chemical formula is identical. These two molecules experience slightly different crystal packing contacts are are expected to be different. Obviously, in general they are very similar (like in your case), unless the crystallization DG is enormous - which means larger that the folding DG. Oliviero On Tue, March 24, 2009 15:49, Sang Hoon Joo wrote: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710
Re: [ccp4bb] OT: Crystallisation compatible detergents
Hi Darren, I'm not aware of any (membrane) protein crystal structures solved with tween20. It's heterogeneous, and its color suggests it contains impurities and/or oxidation products, making it even more heterogenous. It would be better to test the behavior of your complex in the presence of well-defined and often-used detergents such as octylglucoside, (D)DM, LDAO, C8E4 and the like. If this doesn't work only then I'd set up trials with the tween20. Hope this helps, 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: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -Original Message- From: CCP4 bulletin board on behalf of Darren Hart Sent: Tue 3/24/2009 11:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] OT: Crystallisation compatible detergents Hello, We can make a nice 1:1 complex between two proteins that then gradually precipitates during storage at 4 degC. With 0.005% Tween-20 the complex is stable and does not preciptate noticeably. We have used this in buffers to measure the kinetics of binding by Biacore so it is clearly compatible with functionality. Is Tween-20 at this concentration compatible with crystallisation? Is it worth giving a go, or a waste of time? Should we try and remove it, or reprepare the complex in an alternative detergent? If we need to try an alternative, what would people recommend? Thanks in advance for your suggestions, Darren -- ** Dr. Darren Hart, Team Leader High Throughput Protein Lab Grenoble Outstation European Molecular Biology Laboratory (EMBL) ** EMBL webpages: http://tinyurl.com/6xdltl http://tinyurl.com/667jrp Email: h...@embl.fr Tel: +33 4 76 20 77 68 Fax: +33 4 76 20 71 99 Skype: hartdarren Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex 9, France **
Re: [ccp4bb] two identical proteins in one asymmetric unit
Hi Sang Hoon, You should do the refinement in BUSTER, which uses a novel method to impose NCS restraints. These restraints (called LSSR restraints) were designed specifically to provide an answer to your question in a systematic way, by comparing the local environments of corresponding residues in each copy of your protein. Good luck, Joe Jim Fairman a écrit : Sang Hoon, Each molecule in the asymmetric unit is most likely different. I work on a protein that crystallizes as a homodimer with 2 molecules per asymmetric unit and there are some differences between the two (eg: electron density visible for the 14 N-terminal residues in one molecule, but not the other). Cheers, Jim On Tue, Mar 24, 2009 at 11:03 AM, Folmer Fredslund folm...@gmail.com mailto:folm...@gmail.com wrote: Dear Sang They are really different! And I guess you would probably want to use NCS restraints depending on your resolution. Regards, Folmer 2009/3/24 Sang Hoon Joo s...@duke.edu mailto:s...@duke.edu: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710 -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu mailto:jfair...@utk.edu james.fair...@case.edu mailto:james.fair...@case.edu
Re: [ccp4bb] OT: Crystallisation compatible detergents
Dear All, I found this paper quite informative on the procedure for screening and selecting detergents for protein crystallization. http://journals.iucr.org/d/issues/2005/04/00/sx5021/sx5021.pdf Best wishes, Gordon M. Gordon Joyce, Visiting Fellow, Structural Immunology Section, Laboratory of Immunogenetics, NIH/NIAID, 12441 Parklawn Drive, Rockville, MD 20851 Phone: 301 594 0242 Office 301 496 3792 Lab From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Van Den Berg, Bert Sent: Tuesday, March 24, 2009 11:34 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] OT: Crystallisation compatible detergents Hi Darren, I'm not aware of any (membrane) protein crystal structures solved with tween20. It's heterogeneous, and its color suggests it contains impurities and/or oxidation products, making it even more heterogenous. It would be better to test the behavior of your complex in the presence of well-defined and often-used detergents such as octylglucoside, (D)DM, LDAO, C8E4 and the like. If this doesn't work only then I'd set up trials with the tween20. Hope this helps, 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: bert.vandenb...@umassmed.edu http://www.umassmed.edu/pmm/faculty/vandenberg.cfm -Original Message- From: CCP4 bulletin board on behalf of Darren Hart Sent: Tue 3/24/2009 11:22 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] OT: Crystallisation compatible detergents Hello, We can make a nice 1:1 complex between two proteins that then gradually precipitates during storage at 4 degC. With 0.005% Tween-20 the complex is stable and does not preciptate noticeably. We have used this in buffers to measure the kinetics of binding by Biacore so it is clearly compatible with functionality. Is Tween-20 at this concentration compatible with crystallisation? Is it worth giving a go, or a waste of time? Should we try and remove it, or reprepare the complex in an alternative detergent? If we need to try an alternative, what would people recommend? Thanks in advance for your suggestions, Darren -- ** Dr. Darren Hart, Team Leader High Throughput Protein Lab Grenoble Outstation European Molecular Biology Laboratory (EMBL) ** EMBL webpages: http://tinyurl.com/6xdltl http://tinyurl.com/667jrp Email: h...@embl.fr Tel: +33 4 76 20 77 68 Fax: +33 4 76 20 71 99 Skype: hartdarren Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex 9, France **
Re: [ccp4bb] two identical proteins in one asymmetric unit
Sang, They are always different. But depending on your data/parameter ratio, you may be better of assuming they are similar (with NCS restraints) or even identical (with strict NCS). Ask to a friendly crystallographer around you when employing NCS is good for you. Crystallographers with high resolution syndrome are not recommended to use NCS. Certain side effects may include decreased R-Rfree spreads, and local mismatches between electron density and model. Engin P.S. Gerard Kleywegt's papers from 1996 on NCS and refinement practices might be a good place to start reading. Sang Hoon Joo wrote: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD.
Re: [ccp4bb] OT: Crystallisation compatible detergents
Hi Darren I believe that the most frequently used detergent for protein crystallization (not including membrane proteins) is octyl-glucoside. The most important parameter is the CMC of the detergent and the size of the micelles of free detergent if you have micelles around. These considerations do not apply if you are well under the CMC (only free molecules of detergent are present in solution). I believe tween-20 CMC is roughly 0.006% so you are at the border. The problem is you have micelles of detergent is that depending on their size and the MW cutoff of the device you use for protein concentration you may end up concentrating both components protein and detergent and then this is when trouble starts. You may have an unknown amount of concentrated detergent above the CMC, this can be very problematic for crystallization because you may have a lot of phase separation in your drops; phase separation are usually (but not always) not desirable. For membrane protein crystallization we are always facing this problem because we usually work in this regime of critical detergent concentrations. However for soluble proteins that need a little bit of detergent to remain stable this is not as usual. OG (octyl glucoside) is a rather mild detergent with a CMC of about 20 mM (check ANATRACE catalog). To simplify, maybe a little bit too much, the most important is at first to find the minimum amount of detergent you need to keep you sample stable and possibly stay as low under the CMC. And then may be go up in detergent concentration if you don't get the results expected. You can check that your complex stays functional using Biacore (in your specific case) Other detergents very popular for non membrane proteins are DDM (dodecyl maltoside) or CHAPS (zwitterionic cholesterol derivative). So in order of decreasing preference I would suggest OG, CHAPS (which is a totally different type of detergent) and DDM (this one has a very low CMC ~0.15mM I believe, so it is difficult to get rid of it, but it is fairly genlte). An alternative to detergent are non-detergent sulfo-betaines they can sometime have the same protective effect without the trouble of detergents. I hope this helps, Cheers Pascal F. Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics
Re: [ccp4bb] pins - was Re: images
Edward A. Berry wrote: And what about different format of pucks/cassettes/tongs etc? I can still read my old Vax backup tapes (on a linux box with an exabyte tape drive), but my old Yale-style pins won't fit in the Hampton Research cryotongs available at the beamline, they don't sit well on the standard goniometer magnet, and can't be picked up by the automounter. Ed I'm sure we can all agree that standards are important, and this is probably why we all have our own. However, Ed, I can't resist but to point out that the robot in ALS 8.3.1 (Cool Hand Luke or CHL) has a SuperTong that will handle Yale pins as well as every pin Hampton has ever made (24 to 10 mm), and even mods to them like the Syrrx pins (sometimes called ALS pins). Yes, CHL is the $550 robot with the pencils holding the tongs. As for cassettes, we don't use them directly. The sample holder dewar (conveyor) contains 52 magnets identical to those on the goniometer, and you simply unload your cassette or cane or whatever into the conveyor on-site. Best way to do this is via the goniometer itself because then you can center, pre-screen and digitally capture the sample position, run info, etc. for re-centering and data collection during the night. But, it is also possible to load the conveyor from outside the hutch while someone else is collecting data. So, yes, a universal pin solution exists, and the hardware is not expensive, but I gave up a long time ago on trying to get other beamline scientists to expand their support of pin types. But this is not to say that there has not been progress. Aina Cohen I think deserves a lot of credit for managing to generalize support for cassette formats with the UniPuck (which I can at least tell you has gained acceptance at SSRL and ALS), but the UniPuck is by no means universal yet. Something about Europeans and their SPINEs? I think the core reason why beamline staff around the world are not so interested in supporting more standards is not that they are evil or lazy or anything like that but just that they need to prioritize, and most beamlines are still not at the point where they are hunting for new users, but they are trying to hang on to the users they have (especially the ones who pay the bills). -James Holton MAD Scientist
Re: [ccp4bb] OT: Crystallisation compatible detergents
Darren-- Last year I attended the Membrane Protein Crystallization Course organized by Vivian Stojanoff at BNL. James Love talked about screening detergents using ASEC, and we have set this up in our lab now (not as elaborate a system used at the NYCOMPS). We use a universal buffer with a battery of 10-12 detergents and look for nice sharp peaks off the analytical size exclusion column. This has given us good starting points and clearly ruled out some detergents. HTH! annie Annie Hassell Glaxo Smithkline 5 Moore Drive RTP, NC 27709 919/483-3228 919/483-0368 (FAX) annie.m.hass...@gsk.com Darren Hart h...@embl.fr Sent by: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK 24-Mar-2009 11:22 Please respond to Darren Hart h...@embl.fr To CCP4BB@JISCMAIL.AC.UK cc Subject [ccp4bb] OT: Crystallisation compatible detergents Hello, We can make a nice 1:1 complex between two proteins that then gradually precipitates during storage at 4 degC. With 0.005% Tween-20 the complex is stable and does not preciptate noticeably. We have used this in buffers to measure the kinetics of binding by Biacore so it is clearly compatible with functionality. Is Tween-20 at this concentration compatible with crystallisation? Is it worth giving a go, or a waste of time? Should we try and remove it, or reprepare the complex in an alternative detergent? If we need to try an alternative, what would people recommend? Thanks in advance for your suggestions, Darren -- ** Dr. Darren Hart, Team Leader High Throughput Protein Lab Grenoble Outstation European Molecular Biology Laboratory (EMBL) ** EMBL webpages: http://tinyurl.com/6xdltl http://tinyurl.com/667jrp Email: h...@embl.fr Tel: +33 4 76 20 77 68 Fax: +33 4 76 20 71 99 Skype: hartdarren Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex 9, France **
Re: [ccp4bb] pins - was Re: images
So, yes, a universal pin solution exists, and the hardware is not expensive, but I gave up a long time ago on trying to get other beamline scientists to expand their support of pin types. But this is not to say that there has not been progress. Aina Cohen I think deserves a lot of credit for managing to generalize support for cassette formats with the UniPuck (which I can at least tell you has gained acceptance at SSRL and ALS), but the UniPuck is by no means universal yet. Something about Europeans and their SPINEs? We Europeans like our SPINE standard pins but pucks are another matter! I'm kind of glad you brought this up actually because now I can advertise that the Diamond MX beamlines (I02, I03 and I04) will be changing over to using UniPucks from the 1st April. And no, it's not an April's fool joke. Katherine Dr Katherine McAuley, MX Beamline Scientist, Diamond Light Source, UK This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom
Re: [ccp4bb] two identical proteins in one asymmetric unit
I have seen proteins refined as 'the same', modeled to an averaged map etc only to have one of them with much higher Bj because most likely they are NOT the same so watch out by treating them as 'the same' you are losing the very valuable information that you might be looking for Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of ToledoOffice: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu mailto:ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa http://golemxiv.dh.meduohio.edu/~ewa From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Fairman Sent: Tuesday, March 24, 2009 11:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] two identical proteins in one asymmetric unit Sang Hoon, Each molecule in the asymmetric unit is most likely different. I work on a protein that crystallizes as a homodimer with 2 molecules per asymmetric unit and there are some differences between the two (eg: electron density visible for the 14 N-terminal residues in one molecule, but not the other). Cheers, Jim On Tue, Mar 24, 2009 at 11:03 AM, Folmer Fredslund folm...@gmail.com wrote: Dear Sang They are really different! And I guess you would probably want to use NCS restraints depending on your resolution. Regards, Folmer 2009/3/24 Sang Hoon Joo s...@duke.edu: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710 -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
[ccp4bb] Beam Time at LS-CAT Sector-21 at APS
I would like to take the time to welcome all general users to the newest beam lines at the Advance Photon Source, Life Sciences CAT Sector 21. http://ls-cat.org http://protein.nsls.bnl.gov/ There is time available for rapid access, the dates are listed on the website and users can subscribe for upcoming times too. LS-CAT current;y operates three ID end stations, one station (21ID-D) is fully tunable with an MD2, MX-300 and CATS robotic sample changer. 21ID-F G are fixed wavelength at 0.978A with MX-225 and MX-300 detectors, both stations have MD2s and CATS robots too. To schedule time visit the LS-CAT website. If you like to know more send an email: k-bris...@ls-cat.org x6an...@bnl.gov or call: +1 (630) 252 0626 Joseph S Brunzelle, PhD Assistant Research Professor Dept. Mol Pharm Bio Chem Life Sciences Collaborative Access Team Synchrotron Research Center, Northwestern University Ph. 630-252-0629 Fax. 630-252-4664 j-brunze...@northwestern.edu http://www.ls-cat.org
Re: [ccp4bb] two identical proteins in one asymmetric unit
I had a student solve a medium resolution (2.3 A) data set with (unfortunately) 12 identical protein chains in the asymmetric unit. To save a little time, and to take advantage of a large amount of potential averaging we used NCS to do the initial phase of the refinement. For 10 of the 12 chains, everything was hunky-dory. For the 11th and 12th chains, however, there was an extremely messy area of high-sigma difference map density that turned out to be a very interesting ligand-binding interaction. Releasing the symmetry constraints resulted in a very sharp map of the protein chain rearrangement and bound ligand in the two "different" chains. In general, even with homodimers and homotetramers in the ASU, we find that there are often subtle but significant differences in individual protein chains, especially around packing contacts and external loops of the protein. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu Skrzypczak-Jankun, Ewa wrote: I have seen proteins refined as ‘the same’, modeled to an averaged map etc only to have one of them with much higher Bj because most likely they are NOT the same so watch out by treating them as ‘the same’ you are losing the very valuable information that you might be looking for Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of Toledo Office: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jim Fairman Sent: Tuesday, March 24, 2009 11:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] two identical proteins in one asymmetric unit Sang Hoon, Each molecule in the asymmetric unit is most likely different. I work on a protein that crystallizes as a homodimer with 2 molecules per asymmetric unit and there are some differences between the two (eg: electron density visible for the 14 N-terminal residues in one molecule, but not the other). Cheers, Jim On Tue, Mar 24, 2009 at 11:03 AM, Folmer Fredslund folm...@gmail.com wrote: Dear Sang They are really different! And I guess you would probably want to use NCS restraints depending on your resolution. Regards, Folmer 2009/3/24 Sang Hoon Joo s...@duke.edu: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710 -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
[ccp4bb] PDB Submission of SAD data.
I have a mtz from Autosol/resolve that has the following columns: OVERALL FILE STATISTICS for resolution range 0.002 - 0.261 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. Low High label 1 ASC 0 37 0 100.00 18.7 18.7 23.68 1.96 H H 2 NONE 0 25 0 100.00 7.3 7.3 23.68 1.96 H K 3 NONE 0 58 0 100.00 20.5 20.5 23.68 1.96 H L 4 NONE2.3 316.2 0 100.0020.3920.39 23.68 1.96 F FP 5 NONE0.117.2 0 100.00 1.77 1.77 23.68 1.96 Q SIGFP 6 NONE -180.0 180.0 0 100.00 6.2489.14 23.68 1.96 P PHIM 7 NONE 0.000 0.998 0 100.000.2830.283 23.68 1.96 W FOMM 8 NONE -10.911.8 0 100.00 0.01 0.46 23.68 1.96 A HLAM 9 NONE -10.720.6 0 100.00 0.00 0.46 23.68 1.96 A HLBM 10 NONE -4.1 4.5 0 100.00-0.00 0.08 23.68 1.96 A HLCM 11 NONE -3.5 3.1 0 100.00 0.00 0.07 23.68 1.96 A HLDM 12 NONE0.0 1.0 0 100.00 0.10 0.10 23.68 1.96 I FreeR_flag 13 BOTH0.0 0.0 0 100.00 0.00 0.00 23.68 1.96 F FC Should I just prepare my mmcif by pdb_extract_sf -dt F -dp MTZ -c 1 -w 1 -iDAT exptl_fobs_phases_freeR_flags.mtz -o foo.mmcif and submit foo.mmcif to the PDB? Do i need to separately indicate which columns (in my case FP/SIGFP) were used for refinement? Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] two identical proteins in one asymmetric unit
Having dealt with quite a few cases of more than one molecule in the AU (including a couple of dreaded 12-meric assemblies... bleah), I am still looking for the best way to identify proper NCS operators for the myriad of potential combinations of fragments. As has been said, it is generally worthwhile to identify the equivalent portions of the molecules and appropriate NCS weights, not only for potentially finding something interesting in terms of biology, but also for doing the best possible refinement job. I therefore wish there were better tools for this purpose. Overall, I think this area has not received proper attention yet. But it should, because I have the feeling that the impact of a great set of tools would be immense. Eternally hopeful - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Mar 24, 2009, at 1:22 PM, Roger Rowlett wrote: I had a student solve a medium resolution (2.3 A) data set with (unfortunately) 12 identical protein chains in the asymmetric unit. To save a little time, and to take advantage of a large amount of potential averaging we used NCS to do the initial phase of the refinement. For 10 of the 12 chains, everything was hunky-dory. For the 11th and 12th chains, however, there was an extremely messy area of high-sigma difference map density that turned out to be a very interesting ligand-binding interaction. Releasing the symmetry constraints resulted in a very sharp map of the protein chain rearrangement and bound ligand in the two different chains. In general, even with homodimers and homotetramers in the ASU, we find that there are often subtle but significant differences in individual protein chains, especially around packing contacts and external loops of the protein. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu Skrzypczak-Jankun, Ewa wrote: I have seen proteins refined as ‘the same’, modeled to an averaged map etc only to have one of them with much higher Bj because most likely they are NOT the same so watch out by treating them as ‘the same’ you are losing the very valuable information that you might be looking for Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of ToledoOffice: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Fairman Sent: Tuesday, March 24, 2009 11:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] two identical proteins in one asymmetric unit Sang Hoon, Each molecule in the asymmetric unit is most likely different. I work on a protein that crystallizes as a homodimer with 2 molecules per asymmetric unit and there are some differences between the two (eg: electron density visible for the 14 N-terminal residues in one molecule, but not the other). Cheers, Jim On Tue, Mar 24, 2009 at 11:03 AM, Folmer Fredslund folm...@gmail.com wrote: Dear Sang They are really different! And I guess you would probably want to use NCS restraints depending on your resolution. Regards, Folmer 2009/3/24 Sang Hoon Joo s...@duke.edu: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710 -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
[ccp4bb] hkl2000 license
Hi all, I recently collected data at Argonne National Laboratories at BioCars on the 14 IDB beamline. The detector is a mar 165 ccd detector. I was unable to process my datasets at the synchrotron (HKL2000) hoping to process them at home. However, I'm unable to display the frames on HKL2000 mounted on the home computers. I do have the current def.site from biocars downloaded. The error that keeps coming up is No valid license for un-mar165. ERROR CODE 5. We tried getting an additional license (from HKL2000) for the particular detector but it still doesn't seem to work. Any suggestions/help will be greatly appreciated. Thanks very much, Keith Romano Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605
Re: [ccp4bb] two identical proteins in one asymmetric unit
On Tuesday 24 March 2009 12:20:17 Mischa Machius wrote: Having dealt with quite a few cases of more than one molecule in the AU (including a couple of dreaded 12-meric assemblies... bleah), I am still looking for the best way to identify proper NCS operators for the myriad of potential combinations of fragments. As has been said, it is generally worthwhile to identify the equivalent portions of the molecules and appropriate NCS weights, not only for potentially finding something interesting in terms of biology, but also for doing the best possible refinement job. I therefore wish there were better tools for this purpose. Overall, I think this area has not received proper attention yet. But it should, because I have the feeling that the impact of a great set of tools would be immense. The problem can be mitigated enormously by parameterizing the NCS restraints in terms of torsion angles rather than coordinate transforms. This is what shelx does, and it works a fair treat. The point is that is this representation the deviation from NCS caused by an ideal hinge motion shows up only in the handful of torsions corresponding to the hinge point. This means that atoms remote from the hinge point can still adhere closely to the NCS restraint even though there is no single NCS operation that will superimpose them. Ethan Eternally hopeful - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Mar 24, 2009, at 1:22 PM, Roger Rowlett wrote: I had a student solve a medium resolution (2.3 A) data set with (unfortunately) 12 identical protein chains in the asymmetric unit. To save a little time, and to take advantage of a large amount of potential averaging we used NCS to do the initial phase of the refinement. For 10 of the 12 chains, everything was hunky-dory. For the 11th and 12th chains, however, there was an extremely messy area of high-sigma difference map density that turned out to be a very interesting ligand-binding interaction. Releasing the symmetry constraints resulted in a very sharp map of the protein chain rearrangement and bound ligand in the two different chains. In general, even with homodimers and homotetramers in the ASU, we find that there are often subtle but significant differences in individual protein chains, especially around packing contacts and external loops of the protein. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu Skrzypczak-Jankun, Ewa wrote: I have seen proteins refined as ‘the same’, modeled to an averaged map etc only to have one of them with much higher Bj because most likely they are NOT the same so watch out by treating them as ‘the same’ you are losing the very valuable information that you might be looking for Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of ToledoOffice: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Fairman Sent: Tuesday, March 24, 2009 11:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] two identical proteins in one asymmetric unit Sang Hoon, Each molecule in the asymmetric unit is most likely different. I work on a protein that crystallizes as a homodimer with 2 molecules per asymmetric unit and there are some differences between the two (eg: electron density visible for the 14 N-terminal residues in one molecule, but not the other). Cheers, Jim On Tue, Mar 24, 2009 at 11:03 AM, Folmer Fredslund folm...@gmail.com wrote: Dear Sang They are really different! And I guess you would probably want to use NCS restraints depending on your resolution. Regards, Folmer 2009/3/24 Sang Hoon Joo s...@duke.edu: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and
Re: [ccp4bb] PDB Submission of SAD data.
Dear Francis You do not have to tell which columns were used for refinement. The program should pick FP/SIGFP. You can deposit the foo.mmcif and your coordinate to the PDB. The alternative to convert/validate your mtz file is to use the server http://pdb-extract.rcsb.org/auto-check/ . It is easy to use. Also the web page is updated faster than the standard alone program. Regards, Huanwang Francis E Reyes wrote: I have a mtz from Autosol/resolve that has the following columns: OVERALL FILE STATISTICS for resolution range 0.002 - 0.261 === Col SortMinMaxNum % Mean Mean Resolution Type Column num order Missing complete abs. Low High label 1 ASC 0 37 0 100.00 18.7 18.7 23.68 1.96 H H 2 NONE 0 25 0 100.00 7.3 7.3 23.68 1.96 H K 3 NONE 0 58 0 100.00 20.5 20.5 23.68 1.96 H L 4 NONE2.3 316.2 0 100.0020.3920.39 23.68 1.96 F FP 5 NONE0.117.2 0 100.00 1.77 1.77 23.68 1.96 Q SIGFP 6 NONE -180.0 180.0 0 100.00 6.2489.14 23.68 1.96 P PHIM 7 NONE 0.000 0.998 0 100.000.2830.283 23.68 1.96 W FOMM 8 NONE -10.911.8 0 100.00 0.01 0.46 23.68 1.96 A HLAM 9 NONE -10.720.6 0 100.00 0.00 0.46 23.68 1.96 A HLBM 10 NONE -4.1 4.5 0 100.00-0.00 0.08 23.68 1.96 A HLCM 11 NONE -3.5 3.1 0 100.00 0.00 0.07 23.68 1.96 A HLDM 12 NONE0.0 1.0 0 100.00 0.10 0.10 23.68 1.96 I FreeR_flag 13 BOTH0.0 0.0 0 100.00 0.00 0.00 23.68 1.96 F FC Should I just prepare my mmcif by pdb_extract_sf -dt F -dp MTZ -c 1 -w 1 -iDAT exptl_fobs_phases_freeR_flags.mtz -o foo.mmcif and submit foo.mmcif to the PDB? Do i need to separately indicate which columns (in my case FP/SIGFP) were used for refinement? Thanks FR - Francis Reyes M.Sc. 215 UCB University of Colorado at Boulder gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D 8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D -- *Huanwang Yang Ph.D. * * RCSB Protein Data Bank * * Dep. of Chemistry Chemical Biology, Rutgers University * * 610 Taylor Road, Piscataway, NJ 08854-8087 * * Phone: (732)-445-0103 (ext 240); Fax: (732)-445-4320*
Re: [ccp4bb] hkl2000 license
home computers. I do have the current def.site from biocars downloaded. The error that keeps coming up is No valid license for un-mar165. ERROR CODE 5. We tried getting an additional license (from HKL2000) for the particular detector but it still doesn't seem to work. Any suggestions/help will be greatly appreciated. I'd talk to hkl-xray. We've processed data using that hardware (not the same beamline) and were able to see and process the frames using v0.98.695b (not even the latest greatest I think). This is the list of licensed detectors we have for hkl2000: mar Mar345 unsup unsup210 unsup315 un-mar un-mar165 un-brand
Re: [ccp4bb] hkl2000 license
Assuming that you did everything else correctly (e.g. placed the new cr_info file you got from HKL into /usr/local/lib), are you sure you included un-mar165 in your info file? They have this mar option which refers to MAR imaging plate detectors (except MAR345) and thus does not cover CCDs (there were these weird circular MAR imaging plates with, if I am not mistaken, half a minute readout time - were even used at some synchrotron beamlines). Make sure you use un-mar165, if it does not work, you may be better off asking HKL directly. I understand that all the CCD should be encoded un-mar???, so MAR325 CCD (not listed on HKL website) should be requested as un-mar325. And yes, MAR345 is an image plate. On Tue, 2009-03-24 at 15:23 -0400, Keith Romano wrote: Hi all, I recently collected data at Argonne National Laboratories at BioCars on the 14 IDB beamline. The detector is a mar 165 ccd detector. I was unable to process my datasets at the synchrotron (HKL2000) hoping to process them at home. However, I'm unable to display the frames on HKL2000 mounted on the home computers. I do have the current def.site from biocars downloaded. The error that keeps coming up is No valid license for un-mar165. ERROR CODE 5. We tried getting an additional license (from HKL2000) for the particular detector but it still doesn't seem to work. Any suggestions/help will be greatly appreciated. Thanks very much, Keith Romano Department of Biochemistry Molecular Pharmacology 970L Lazare Research Building University of Massachusetts Medical School 364 Plantation Street Worcester, MA 01605 -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] two identical proteins in one asymmetric unit
My favorite trick was to define domain-wise ncs restraints, extensively minimize with and without them, then plot the difference in real-space R factor per residue. Ones that jump up when restrained are usually involved in crystal packing, etc, and should be removed from the restraints. In my clumsy hands, this required 4 cns scripts followed by importing the real-space R lists into excel. There must be a better way? Phoebe Original message Date: Tue, 24 Mar 2009 14:20:17 -0500 From: Mischa Machius mischa.mach...@utsouthwestern.edu Subject: Re: [ccp4bb] two identical proteins in one asymmetric unit To: CCP4BB@JISCMAIL.AC.UK Having dealt with quite a few cases of more than one molecule in the AU (including a couple of dreaded 12-meric assemblies... bleah), I am still looking for the best way to identify proper NCS operators for the myriad of potential combinations of fragments. As has been said, it is generally worthwhile to identify the equivalent portions of the molecules and appropriate NCS weights, not only for potentially finding something interesting in terms of biology, but also for doing the best possible refinement job. I therefore wish there were better tools for this purpose. Overall, I think this area has not received proper attention yet. But it should, because I have the feeling that the impact of a great set of tools would be immense. Eternally hopeful - MM Mischa Machius, PhD Associate Professor Department of Biochemistry UT Southwestern Medical Center at Dallas 5323 Harry Hines Blvd.; ND10.214A Dallas, TX 75390-8816; U.S.A. Tel: +1 214 645 6381 Fax: +1 214 645 6353 On Mar 24, 2009, at 1:22 PM, Roger Rowlett wrote: I had a student solve a medium resolution (2.3 A) data set with (unfortunately) 12 identical protein chains in the asymmetric unit. To save a little time, and to take advantage of a large amount of potential averaging we used NCS to do the initial phase of the refinement. For 10 of the 12 chains, everything was hunky-dory. For the 11th and 12th chains, however, there was an extremely messy area of high-sigma difference map density that turned out to be a very interesting ligand-binding interaction. Releasing the symmetry constraints resulted in a very sharp map of the protein chain rearrangement and bound ligand in the two different chains. In general, even with homodimers and homotetramers in the ASU, we find that there are often subtle but significant differences in individual protein chains, especially around packing contacts and external loops of the protein. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu Skrzypczak-Jankun, Ewa wrote: I have seen proteins refined as ‘the same’, modeled to an averaged map etc only to have one of them with much higher Bj because most likely they are NOT the same so watch out by treating them as ‘the same’ you are losing the very valuable information that you might be looking for Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of Toledo Office: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Fairman Sent: Tuesday, March 24, 2009 11:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] two identical proteins in one asymmetric unit Sang Hoon, Each molecule in the asymmetric unit is most likely different. I work on a protein that crystallizes as a homodimer with 2 molecules per asymmetric unit and there are some differences between the two (eg: electron density visible for the 14 N-terminal
Re: [ccp4bb] two identical proteins in one asymmetric unit
Hello Roger, did you publish your results? Your case sounds very educational and might be interesting for teaching purposes, so a reference would be a nice thing to have. Tim -- Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A On Tue, 24 Mar 2009, Roger Rowlett wrote: I had a student solve a medium resolution (2.3 A) data set with (unfortunately) 12 identical protein chains in the asymmetric unit. To save a little time, and to take advantage of a large amount of potential averaging we used NCS to do the initial phase of the refinement. For 10 of the 12 chains, everything was hunky-dory. For the 11th and 12th chains, however, there was an extremely messy area of high-sigma difference map density that turned out to be a very interesting ligand-binding interaction. Releasing the symmetry constraints resulted in a very sharp map of the protein chain rearrangement and bound ligand in the two different chains. In general, even with homodimers and homotetramers in the ASU, we find that there are often subtle but significant differences in individual protein chains, especially around packing contacts and external loops of the protein. Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu Skrzypczak-Jankun, Ewa wrote: I have seen proteins refined as ?the same?, modeled to an averaged map etc only to have one of them with much higher Bj because most likely they are NOT the same so watch out by treating them as ?the same? you are losing the very valuable information that you might be looking for Ewa Dr Ewa Skrzypczak-Jankun Associate Professor University of Toledo Office: Dowling Hall r.2257 Health Science Campus Phone: 419-383-5414 Urology Department Mail Stop #1091 Fax: 419-383-3785 3000 Arlington Ave. e-mail: ewa.skrzypczak-jan...@utoledo.edu Toledo OH 43614-2598 web: http://golemxiv.dh.meduohio.edu/~ewa From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jim Fairman Sent: Tuesday, March 24, 2009 11:25 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] two identical proteins in one asymmetric unit Sang Hoon, Each molecule in the asymmetric unit is most likely different. I work on a protein that crystallizes as a homodimer with 2 molecules per asymmetric unit and there are some differences between the two (eg: electron density visible for the 14 N-terminal residues in one molecule, but not the other). Cheers, Jim On Tue, Mar 24, 2009 at 11:03 AM, Folmer Fredslund folm...@gmail.com wrote: Dear Sang They are really different! And I guess you would probably want to use NCS restraints depending on your resolution. Regards, Folmer 2009/3/24 Sang Hoon Joo s...@duke.edu: I am refining my crystal structure in which I have two identical chains in one asymmetric unit. Space group is H32 and each chain yields me a biological trimer as expected. The problem is, do I have to assume they are identical, or they are really different. After each cycle of refinement, if I try to align two molecules I get ~ 0.17 RMSD. -- Sang Hoon Joo, PhD Postdoctoral Associate Duke University 239 Nanaline H. Duke Box 3711, DUMC Durham, NC 27710 -- Jim Fairman Graduate Research Assistant Department of Biochemistry, Cellular, and Molecular Biology (BCMB) University of Tennessee -- Knoxville 216-368-3337 jfair...@utk.edu james.fair...@case.edu
[ccp4bb] Postdoc Crystallographer Position - Berkeley
Lawrence Berkeley Laboratory Postdoctoral Researcher Job ID: 22539 Division: Physical Biosciences Date Opened: 3/24/2009 Summary: A postdoctoral position is immediately available to study structure and function of macromolecular complexes and enzymes using X-ray crystallographic methods. This position is joint between the Center for Protein Folding Machinery (www.proteinfoldingcenter.org) and the Joint BioEnergy Institute (www.jbei.org). Robotic hardware for performing crystallization trials and imaging results are available. Crystals will be characterized and data collected using the beamline resources of the Berkeley Center for Structural Biology at the Advanced Light Source. The biological systems to be studied are: - Type-II chaperonins responsible for the refolding of unfolded proteins in archeal and mammalian cells. - Glycosyl transferases responsible for the synthesis of hemicellulose in plants. - Novel glycosyl hydrolases that are able to breakdown cellulosic material for biofuels production. Duties: Using robotic hardware to perform screens for crystallization conditions. Optimization of crystallization conditions. Biophysical characterization of protein samples using standard techniques, such as dynamic light scattering. Characterization of crystals using the Berkeley Center for Structural Biology beamlines at the Advanced Light Source. Collection and analysis of diffraction data, model building and structure refinement. Extensive email and verbal interaction with other researchers. Qualifications: Ph.D. or equivalent in a scientific discipline, preferably structural biology, biology, or chemistry. Demonstrated experience of scientific research using crystallographic methods. Experience with crystallization methods. Solid interpersonal skills and the ability to work in a team environment are critical. Ability to communicate with a broad range of researchers. Note that this is a one-year appointment with renewal based on performance and continued funding. Lawrence Berkeley National Laboratory is a world leader in science and engineering research, with 11 Nobel Prize recipients over the past 75 years, and 59 present members of the National Academy of Sciences. LBNL conducts unclassified research across a wide range of scientific disciplines and hosts four national user facilities. AA/EEO employer committed to the development of a safe and diverse workforce. Learn more at http://www.lbl.gov. To apply visit http://cjo.lbl.gov/ and search for the job number 22539 or visit: http://jobs.lbl.gov/LBNLCareers/details.asp?jid=22539p=1 -- Paul Adams Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab Adjunct Professor, Department of Bioengineering, U.C. Berkeley Vice President for Technology, the Joint BioEnergy Institute Head, Berkeley Center for Structural Biology Building 64, Room 248 Tel: 1-510-486-4225, Fax: 1-510-486-5909 http://cci.lbl.gov/paul Lawrence Berkeley Laboratory 1 Cyclotron Road BLDG 64R0121 Berkeley, CA 94720, USA. Executive Assistant: Patty Jimenez [ pajime...@lbl.gov ] [ 1-510-486-7963 ] --
