Re: [ccp4bb] To scale or not to scale: XDS_ASCII.HKL input to POINTLESS/AIMLESS
Dear Graeme, some points for further clarification- The CORRECT corrections you mention all depend on the geometric description of the experiment. This geometric description of the experiment is refined by CORRECT, to come up with accurate values for a) application of polarization correction (which you were mentioning) b) application of the zeta factor which has to do with the the lengthening of the path of a reflection passing through the Ewald sphere at an angle c) intensity correction depending on finite detector thickness, detector material, wavelength, and angle (you were also mentioning this; it is the 1. item in the SILICON article in XDSwiki) d) positions of reflections on the surface of the detector. This is the second item in the SILICON article in XDSwiki. See also http://xds.mpimf-heidelberg.mpg.de/html_doc/xds_parameters.html#SILICON= e) air absorption (http://xds.mpimf-heidelberg.mpg.de/html_doc/xds_parameters.html#AIR=) Maybe I forgot something, but this may complete the picture somewhat. best, Kay On Mon, 17 Nov 2014 09:44:13 +, Graeme Winter graeme.win...@gmail.com wrote: Dear Nukri, The following is my opinion which I think is worth discussion, and are based on my understanding of what XDS does in the CORRECT step. Firstly, I tend to find the global refinement in the CORRECT step useful for getting a good unit cell recycling the orientation matrix etc. for reintegration. This is not related to scaling, but is useful, e.g.: http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Optimisation#Re-INTEGRATEing_with_the_correct_spacegroup.2C_refined_geometry_and_fine-slicing_of_profiles More relevant to the intensities: in integration the LP correction is calculated assuming an unpolarized beam - if the data are from a synchrotron these need to be corrected again for the correct polarization - something which the correct step does (obviously given this on the command-line). Pointless will also do this but assumes unless given a correct value that the beam is quite polarized. Mostly: care needs to be taken, particularly if using a wavelength which may be confused with a lab source... I also understand that the XDS CORRECT step applies a DQE correction for Pilatus data, taking into account the geometry of the experiment, the sensor thickness photon energy. If you have a two theta offset and are using relatively high energy (say 14 keV or so?) then this may have odd effects on your data. At detector two theta = 0 this is less of a problem. This can be a gotcha with processing small molecule data recorded with a little Pilatus. Best wishes Graeme On Fri Nov 14 2014 at 6:15:31 PM Sanishvili, Ruslan rsanishv...@anl.gov wrote: Dear Graeme, Could you elaborate on There are also some subtleties to making (b) work properly... some more? I have a feeling, from observing the beamline users, that many choose to use this option. It would be very helpful for them to know what are those subtleties and how to best make it work properly. Many thanks, Nukri Ruslan Sanishvili (Nukri) Macromolecular Crystallographer GM/CA@APS X-ray Science Division, ANL 9700 S. Cass Ave. Lemont, IL 60439 Tel: (630)252-0665 Fax: (630)252-0667 rsanishv...@anl.gov -- *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Graeme Winter [graeme.win...@gmail.com] *Sent:* Thursday, November 13, 2014 2:15 AM *To:* CCP4BB@JISCMAIL.AC.UK *Subject:* Re: [ccp4bb] To scale or not to scale: XDS_ASCII.HKL input to POINTLESS/AIMLESS Dear Kay Just to comment on (e) since you say you don't know why anyone would want to do this, yet this is exactly what xia2 -3d does :o) I use AIMLESS to merge data already scaled by XDS CORRECT or XSCALE as a way to get a report on the merging statistics which includes all of the AIMLESS analysis, and to generate harvesting files for deposition. Like you, I look forward to studies of (a) - (e) think of all of these (c) is by far the worst idea, from gut instinct. There are also some subtleties to making (b) work properly... For anyone who has time on their hands would like to do this study, be sure to consider a range of crystal symmetries as it is possible that some strategies which are safe in PG 422 (say) are not in PG 2. Best wishes Graeme On Wed Nov 12 2014 at 10:07:10 PM Kay Diederichs kay.diederi...@uni-konstanz.de wrote: Hi Wolfram, it took me a while until I realized that you mean overfitting when you said o-word. You can abuse XDS in a number of ways, and I would call them overfitting the data although that would be using the word in a somewhat strained way: reducing WFAC1 below 1, decreasing REFLECTIONS/CORRECTION_FACTOR below 50 come to mind, but in an extended sense there are other ways: rejecting frames for no other reason than that they have low I/sigma or high Rmeas, ... People always seem to find ways to beautify their precision indicators, but
[ccp4bb] Empty CORRECT.LP file
Hello, I have a question regarding the XDS output file CORRECT.LP. I have a dataset collected on a Pilatus which has 4960 frames. XDS ran over night to process the data with the ALL job activated and terminated successfully but the output CORRECT.LP file contains ZERO bytes, no data. I ran XDS on a MAC. Was something excluded in the XDS.INP file? Hope to get some feed back. Regards, Chris Browning This email message and any attachments are confidential and intended for use by the addressee(s) only. If you are not the intended recipient, please notify me immediately by replying to this message, and destroy all copies of this message and any attachments. Thank you.
Re: [ccp4bb] Empty CORRECT.LP file
Dear Chris, you can replace the JOB=ALL with JOB=CORRECT and rerun xds. It won't take long and this way you can check of CORRECT.LP is written correctly or if you receive an error message (your partition might be full or your disk quota exceeded). Regards, Tim On 11/18/2014 10:42 AM, Christopher Browning wrote: Hello, I have a question regarding the XDS output file CORRECT.LP. I have a dataset collected on a Pilatus which has 4960 frames. XDS ran over night to process the data with the ALL job activated and terminated successfully but the output CORRECT.LP file contains ZERO bytes, no data. I ran XDS on a MAC. Was something excluded in the XDS.INP file? Hope to get some feed back. Regards, Chris Browning This email message and any attachments are confidential and intended for use by the addressee(s) only. If you are not the intended recipient, please notify me immediately by replying to this message, and destroy all copies of this message and any attachments. Thank you. -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A signature.asc Description: OpenPGP digital signature
[ccp4bb] Post-doc position - membrane protein complex
A candidate is thought for a two-year position (an extension is possible) to work on structural characterisation of respiratory complex I from T. thermophilus. Complex I is central to bioenergetics – it is the first and largest enzyme of the respiratory chain in mitochondria and bacteria. It is also involved in a wide range of human diseases. We use the bacterial enzyme as a ‘minimal’ model of the more elaborate mammalian complex I. We have determined, by X-ray crystallography, all currently known atomic structures of complex I, starting with the hydrophilic domain (Science 2005, 2006), followed by the membrane domain (Nature 2010, 2011) and, finally, the recent (Nature 2013) structure of the entire Thermus thermophilus complex (536 kDa, 16 subunits, 9 Fe-S clusters, 64 TM helices). Structures suggest a unique mechanism of coupling between electron transfer in the hydrophilic domain and proton translocation in the membrane domain, via long-range (up to ~200 Å) conformational changes. In order to determine the intriguing coupling mechanism, we are now working on the structures of the complex in different redox states, with various bound substrates. We are also interested in establishing the mode of action of various inhibitors of complex I. The post holder will be involved in taking this challenging project further, which requires extensive experience in protein purification and X-ray crystallography, preferably acquired working with membrane proteins or macromolecular assemblies. The post is partly funded via collaboration with Pharma. Initially several months will be spent training in the MRC Mitochondrial Biology Unit in Cambridge, UK and then the project will be moved together with the rest of the group to the Institute of Science and Technology in Austria, near Vienna. IST Austria is a new institute dedicated to basic research, providing world-class research environment and benefits. We are looking to fill the post by early 2015. All queries and applications with CVs and names of 2-3 academic referees should be forwarded to Dr. Leonid Sazanov (saza...@mrc-mbu.cam.ac.uk).
Re: [ccp4bb] molprobity clashscore, symmetry-related molecules?
Hi Tim, While we did not make this information available yet, there is a way to get non-bonded clash score that does take into account symmetry related clashes using the Phenix command: phenix.pdb_interpretation .pdb nonbonded_clashscore=True Testing for clashes is done in a slightly different method than the one in MolProbity, so the clash score for clashes not due to symmetry will not be exactly the same as the clash score produced by MolProbity. So for example running from the command prompt phenix.fetch_pdb 1d11 phenix.pdb_interpretation 1d11.pdb nonbonded_clashscore=True will produce a report ending with: Nonbonded clashscore Without symmetry operation and solvent-solvent clashes: 12.35 Due to symmetry operation: 32.92 Solvent-solvent: 8.23 Total: 53.50 Thanks
[ccp4bb] Plasmid for cloning scFV into Fab format and Bacterial expression for crystallography
Hi everyone, I have a few single chain antibodies (scFV) that I would like to express in a Fab format in bacteria for crystallization purposes. Could you suggest some plasmids that have success in such kind of projects? Are there commercial plasmids consisting of antibody constant regions ready for bacterial expression, where I can just clone my scFv and it would be ready for Fab expression (probably periplasmic or secreted in supernatant) If commercial plasmids are not available, could you suggest where I could request them from? Thanks in advance, Ivan
[ccp4bb] how to combine two derivative datasets that are not isomorphous?
Hi All, I have two derivative datasets (heavy atom A and B) and two native datasets (a and b), A and a are isomorphous, B and b are isomorphous, however, a and b (or A and B) are not isomorphous. I was able to make two difference patterson maps (FA-Fa and FB-Fb) and search for heavy atoms against them, the possible positions of heavy atom A and B are very close to each other in the unit cell (which seems to me that I am very close to a right phase, though not there yet), I am wondering if there is any means for me to combine the information from FA-Fa and FB-Fb as the two native datasets are not isomorphous? And also, I have a homology model which I tried molecular replacement and failed, is there any means for me to combine the information from the model too? Best, Bei
Re: [ccp4bb] Plasmid for cloning scFV into Fab format and Bacterial expression for crystallography
Hi Ivan, Just have a look at this paper: Skerra, A. (1994) A general vector, pASK84, for cloning, bacterial production, and single-step purification of antibody Fab fragments. Gene 141, 79-84. We (and other labs) have used this plasmid as well as its successors for the cloning and preparation of numerous recombinant Fabs, and a couple of them have also been crystallized successfully (some X-ray structures still awaiting publication): Schiweck, W. Skerra, A. (1995) Fermenter production of an artificial Fab fragment, rationally designed for the antigen cystatin, and its optimized crystallization through constant domain shuffling. Proteins: Struct. Funct. Genet. 23, 561-565. I will be happy to share these plasmids for academic research purposes. Best regards, Arne Skerra Am 18.11.2014 um 19:39 schrieb xaravich ivan xaravich.i...@gmail.com: Hi everyone, I have a few single chain antibodies (scFV) that I would like to express in a Fab format in bacteria for crystallization purposes. Could you suggest some plasmids that have success in such kind of projects? Are there commercial plasmids consisting of antibody constant regions ready for bacterial expression, where I can just clone my scFv and it would be ready for Fab expression (probably periplasmic or secreted in supernatant) If commercial plasmids are not available, could you suggest where I could request them from? Thanks in advance, Ivan Prof. Dr. Arne Skerra Lehrstuhl f. Biologische Chemie | Technische Universitaet Muenchen Emil-Erlenmeyer-Forum 5 | 85350 Freising-Weihenstephan | Germany Phone: +49 (0)8161 71-4351 | Fax: -4352 eMail: ske...@tum.de | http://www.wzw.tum.de/bc
Re: [ccp4bb] how to combine two derivative datasets that are not isomorphous?
You don't mention any quality indicators on your derivatives, nor resolution. Presuming they actually have some decent phasing power, you may be able to generate phases maps using SIR phasing + solvent flattening. If you can do that for each isomorphous set, then you could combine them using multiple space group noncrystallographic averaging. Or, if the derivatives contain atoms with anomalous scattering, you could solve a derivative alone with SAD methods, or a derivative + native set with SIRAS. Once you get crude maps, you can once again try to combine the two sets with multiple space group averaging. As for MR, there are lots of things you can try. Once again, you have provided no detail on what the sequence similarity is, or any other factor that would allow us to judge the likelihood of success. You could search again with the model clipped down to poly-Ala. You could search again with any external loops trimmed off. If it is a multiple domain molecule, you could search individually with single domains. You could find additional search models, and search with a suite rather than single model. MR programs will suck up as much free time as you can provide them. On 11/18/14 15:01, joy yang wrote: Hi All, I have two derivative datasets (heavy atom A and B) and two native datasets (a and b), A and a are isomorphous, B and b are isomorphous, however, a and b (or A and B) are not isomorphous. I was able to make two difference patterson maps (FA-Fa and FB-Fb) and search for heavy atoms against them, the possible positions of heavy atom A and B are very close to each other in the unit cell (which seems to me that I am very close to a right phase, though not there yet), I am wondering if there is any means for me to combine the information from FA-Fa and FB-Fb as the two native datasets are not isomorphous? And also, I have a homology model which I tried molecular replacement and failed, is there any means for me to combine the information from the model too? Best, Bei -- === All Things Serve the Beam === David J. Schuller modern man in a post-modern world MacCHESS, Cornell University schul...@cornell.edu
[ccp4bb] superpose bug?
Hi: I have a bunch of pdb files and would like to superpose them to one of them based on secondary structure matching. I would like to get a script to do so (not a one-by-one job by hand). I tried superpose but it seemed to have some problems with output option. Assuming C.pdb is the output pdb, and A.pdb and B.pdb are inputs. superpose A.pdb B.pdb (this works, without output) superpose A.pdb B.pdb C.pdb (* ERROR #15 READ:) Adding -s option get same errors (without output pdb it works). When running within ccp4i with output C.pdb, it does work. I tried to generate an input script from ccp4i running. Unfortunately, running with the particular superpose using S-S-M option did not generate an input card (other options OK, what a bad luck). I appreciate a working script (ccp46.4.0) or a debugged source code for compilation. Thanks. Lijun Liu
[ccp4bb] Fwd: [ccp4bb] B-factor blurring
My sincere thanks to all who are responding to my request below. To be explicit, my question relates to B-factor blurring (+B correction), not to B-factor sharpening (-B correction). thanks Mike Begin forwarded message: From: Mike Lawrence lawre...@wehi.edu.au Subject: [ccp4bb] B-factor blurring Date: 18 November 2014 12:01:07 pm AEDT To: CCP4BB@JISCMAIL.AC.UK Reply-To: Mike Lawrence lawre...@wehi.edu.au Dear all can anyone help me with literatures references to B-factor blurring as a technique to reveal low resolution features in an electron density map? I have seen the poster from Andrea Thorn at http://shelx.uni-ac.gwdg.de/~athorn/pdf/thorn_iucr2014_poster.pdf but was wondering if there were any alternative references? with many thanks! Mike __ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. __
[ccp4bb] Postdoctoral position at Houston, TX, USA
Dear colleagues, There is an open postdoctoral position in an infectious disease lab at Houston Methodist Hospital Research Institute. Please see the announcement below. Postdoctoral Fellow position - Houston Methodist Research Institute (HMRI) We are seeking a structural biologist/biochemist to join the Department of Pathology and Genomic Medicine at Houston Methodist Hospital Research Institute, Houston, TX, USA. We employ a multidisciplinary approach to dissect the mechanisms of bacterial signaling pathways and understand the implications of such signaling pathways in the bacterial pathogenesis. We use a variety of genetic, biochemical, biophysical, structural, and mouse infection methods to identify the novel cellular processes with the long-term goal of targeting these processes for antibiotic development. Some of the key areas we are currently investigating include the bacterial signaling systems, and the bacterial sensory system for metal and carbohydrate acquisition. The group uses a wide range of techniques that include X-ray crystallography, SAXS, bacterial genetics, biochemical and transcriptome methods, and animal infection studies. Qualifications and Experience Qualified candidates will have (or be about to obtain) a Ph.D. degree. The candidate is expected to have experience in protein chemistry and crystallography. Ideally, the candidate will be familiar with biochemical techniques and analyses. The ability to work in a team, good organizational and communication skills in English as well as initiative, flexibility and good learning ability are also desired. For more information, please contact: mkumarasw...@houstonmethodist.org
[ccp4bb]
Gesendet:Dienstag, 18. November 2014 um 18:34 Uhr Von:Mike Lawrence lawre...@wehi.edu.au An:CCP4BB@JISCMAIL.AC.UK Betreff:[ccp4bb] Fwd: [ccp4bb] B-factor blurring My sincere thanks to all who are responding to my request below. To be explicit, my question relates to B-factor blurring (+B correction), not to B-factor sharpening (-B correction). thanks Mike Begin forwarded message: From: Mike Lawrence lawre...@wehi.edu.au Subject: [ccp4bb] B-factor blurring Date: 18 November 2014 12:01:07 pm AEDT To: CCP4BB@JISCMAIL.AC.UK Reply-To: Mike Lawrence lawre...@wehi.edu.au Dear all can anyone help me with literatures references to B-factor blurring as a technique to reveal low resolution features in an electron density map? I have seen the poster from Andrea Thorn at http://shelx.uni-ac.gwdg.de/~athorn/pdf/thorn_iucr2014_poster.pdf but was wondering if there were any alternative references? with many thanks! Mike __ The information in this email is confidential and intended solely for the addressee. You must not disclose, forward, print or use it without the permission of the sender. __
[ccp4bb] Water molecules after refinement
Dear All, I am sorry to ask this simple question, but I really need suggestions for this. As the refinement has been done at 3.0 Angstrom after refinement the water molecules were added by using find waters in coot. After adding water refinement was done using Refmac 05. Now when I look on the added water molecules some has density around it and some does not (even no red density around it). Should I remove later water molecules. and again do the refinement ? Please give some explanation also for this. as the R free and R meas has reduced to some extent, also the r m s d contour of map level was kept at 1.09 after refinement. I welcome your suggestions, thanks in advance. Regards Jeorge
Re: [ccp4bb] Water molecules after refinement
Dear Jeorge are the water molecules without electron density properly coordinated? These could be ghosts... Additionally, did you solve the structure by molecular replacement? If so, in your search model, do you see these water molecules at the same / nearby location? Again, those could be model bias. Finally, depending on your resolution, you can expect a certain amount of water molecule (depends on other factors, obviously). If you find 'too many' (difficult to define 'too many'), it might be one indication that you are over-refining things. I know I did not answer your question, but hope this could help. Others will have better comments I guess. Cheers, Leo On 19 Nov 2014, at 07:33, jeorgemarley thomas kirtswab...@gmail.com wrote: Dear All, I am sorry to ask this simple question, but I really need suggestions for this. As the refinement has been done at 3.0 Angstrom after refinement the water molecules were added by using find waters in coot. After adding water refinement was done using Refmac 05. Now when I look on the added water molecules some has density around it and some does not (even no red density around it). Should I remove later water molecules. and again do the refinement ? Please give some explanation also for this. as the R free and R meas has reduced to some extent, also the r m s d contour of map level was kept at 1.09 after refinement. I welcome your suggestions, thanks in advance. Regards Jeorge
