[ccp4bb] Off-Topic_Call for proposals utilising Instruct-funded structural biology techniques
Dear All A call is now open for free access to some of the best structural biology facilities in Europe. Instruct has 16 Instruct Centres around Europe and in Israel offering access to a full range of advanced technologies for obtaining structural and dynamic information on systems in various dimensions, complexity and resolution. Instruct also ensures expert support at the facility for the best research outcome. Access to the Instruct Centre technologies is FREE to scientists from Instruct member countries. Academic students and scientists who are working in laboratories of any of the Instruct member countries (presently BE, CZ, ES, FR, DE, IL, IT, NL, PT, SE, UK) are eligible to apply for access. Access is free and we pay your travel and consumables. Proposals should outline an integrated programme of research that requires one or more Instruct technologies to achieve the aims of the project, and which are not ordinarily available in your own institution or through national facilities. All proposals are evaluated quickly on scientific merit. Proposals for funded access to the full set of Instruct facilities are now invited. Register on the Instruct website (http://www.structuralbiology.eu/) and submit your proposal online at http://www.structuralbiology.eu/proposal/submit. Further information is available on the Instruct website (HomeResourcesProposal Submission). The deadline for this round of proposal submission is 5pm CET, Friday 11th April 2014. Best wishes Claudia Dr Claudia Alen Amaro Scientific Project Manager Instruct: An Integrated Structural Biology Infrastructure for Europe, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Headington OX3 7BN, UK Tel: +44 1865 287808 email: clau...@strubi.ox.ac.uk Follow us on twitter @instructhub
[ccp4bb] On Behalf Of Katherine Sippel, Re: [ccp4bb] High Salt Cryo
Hi, what was not mentioned in the ccp4 discussion is The use of trimethylamine N-oxide as a primary precipitating agent and related methylamine osmolytes as cryoprotective agents for macromolecular crystallography Acta Cryst. (2012). D68, 69-81 I found it as a very potent compound for cryo-preservation of crystals. Kind regards, Reiner -- Dr. Reiner Kiefersauer research scientist Proteros Biostructures GmbH Bunsenstraße 7a D-82152 Planegg-Martinsried Germany Tel.: +49 (0) 89 700761 -0 Fax: +49 (0) 89 700761 -15 E-mail: kiefersa...@proteros.demailto:kiefersa...@proteros.de Die in dieser e-mail beinhaltete Information unterliegt der Geheimhaltung. Sie ist ausschließlich für den genannten Empfänger bestimmt. Falls Sie nicht der genannte Empfänger sind, bitten wir Sie, uns darüber in Kenntnis zu setzen und alle bestehenden Kopien zu vernichten. This message and any attached files contain privileged and/or confidential information intended only for the use of the addressee. If you are not the addressee, or the person responsible for delivering it to the person addressed, you may not read, use, copy or deliver this to anyone else. If you received this message by mistake, please immediately notify us and delete it from your system. All care has to be taken to ensure this message and any attachments are virus free. No responsibility is accepted for any virus infections caused by the receipt of this message. Geschäftsführer: Dr. Torsten Neuefeind, Arnd Christ Amtsgericht München HRB-Nr. 121 476 - VAT-ID: DE 195 857 418 - Steuernummer 143/173/20265
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
Jacob is right, there definitely seem to be problems with the data. Perhaps you and your supervisor should consider contacting privately the developers of data processing programs that have participated in the thread like Kay and Harry (and others perhaps too) to try and get the best out of your data. There is a limit to what refinement programs can do when there is a real problem in the data which is not taken care of properly. My 2p thoughts. Boaz Boaz Shaanan, Ph.D. Dept. of Life Sciences Ben-Gurion University of the Negev Beer-Sheva 84105 Israel E-mail: bshaa...@bgu.ac.il Phone: 972-8-647-2220Skype: boaz.shaanan Fax: 972-8-647-2992 or 972-8-646-1710 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Chris Fage [cdf...@gmail.com] Sent: Monday, February 24, 2014 12:52 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] High Rwork/Rfree vs. Resolution Thanks again for the advice, everyone. As suggested, I tried NCS and TLS in phenix.refine, although my R-factors did not budge. I am now giving PDB_REDO and simulating annealing in PHENIX a shot. I am also looking into setting up XDS. Forgive my ignorance, but I am not sure how to check whether the bulk solvent model is reasonable. For these crystals, HKL2000 does invariably report high mosaicity along one axis (it is in the red). Yes, the structure was solved by MR. For the 1.65-angstrom map, the model is very complete, with density missing only for the N-terminal 6xHis tag and first three residues, as well as 5-10 other residues on flexible loops (the protein is ~300 residues, including the tag). Most side chains are well resolved. The quality of the 1.90-angstrom map is lower, with more gaps, more noise, and less side-chain coverage. In each map, there is no remaining density that legitimately needs to be filled. I have attached representative frames and relevant details from the HKL2000 scale logs. (Note that the 1.65-A set was originally scaled to 1.53 A.) As for making the datasets available before publication, I would have to check with my supervisor. The idea might not fly with him, as the structure is expected to be of relatively high impact. Best, Chris On Sat, Feb 22, 2014 at 3:00 AM, Francis Reyes francis.re...@colorado.edu wrote: I'm guessing the low completeness of the 1.65 angstrom dataset has to do with obstacles the processing software encountered on a sizable wedge of frames (there were swaths of in red in HKL2000). I'm not sure why this dataset in particular was less complete than the others. This is bad. Large swaths of red circles during integration is bad. I believe (check the Denzo manual) this means overlaps and overlaps get thrown out. Thus you are getting lower completeness. Was your oscillation range too large? Crystal very mosaic? However this could be because of a poor crystal orientation matrix by HKL2000 which in some cases can be alleviated by mosflm and xds. (HKL2000 is much more manual, there's a lot of buttons, which means you can shoot yourself in the foot if you are not careful). I would be particularly interested in a resolution bin breakdown in the integration and merging statistics. (I/sig and rmerge). You might as well post the refinement statistics (r and rfree) by resolution bin as well. You have a smallish unit cell that shoots to high resolution and getting a reasonable completion of the low resolution bins is paramount. Post the completeness of the 20-10A bin. Is this molecular replacement? How complete is the model? Aside from the completeness of the model, how far is it from the target? You mentioned that some regions of your crystal had smeary spots. This is also bad, particularly if the errors are not random (I.e anisotropic along one axis). This will confuse ML refinement. Let's see a single frame of your data. Cheers, F
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
(Off-list) Dear Kay and Harry, You mentioned difficult datasets being of interest for the ACA conference. I am not coming, but do have some interesting datasets, viz., many datasets of a particular crystal form of a calmodulin/peptide complex which have defied my [not exhaustive] attempts to solve them. I have a closely-related complex structure which was fairly easy to solve, so I am not so invested in working super hard at the difficult one, but all the same, would really like to see what it looks like. The crystals have calcium and sulfur atoms (some collected at 1.6, some at 1.0 Ang) and there is also the plethora of possible MR models in the pdb and my closely-related complex, in which calmodulin looks dissimilar from the other pdb cam structures, based on DALI. I think these particular datasets may be tetartohedrally twinned, but from the literature it seems this can be overcome by tweaking the right MR model, maybe using some anomalous, etc. Interested? I would give you all the images, I guess, which would be ~10 sets of varying number of frames? Radiation damage appears to be of variable degree. Jacob -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Kay Diederichs Sent: Sunday, February 23, 2014 2:55 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] High Rwork/Rfree vs. Resolution Projects and problems like this are clearly a justification for asking to deposit not only the results from data processing, but also the raw data frames. These would allow developers to improve the models underlying their algorithms, and to find those corner cases where the algorithms break. That would help everyone. Maybe you could make this dataset (and sequence) available for the forthcoming IUCr conference, as an example for a difficult dataset? (send email to Ed Collins or me) You would profit from the fact that experienced crystallographers do their best to make the most of your data. best, Kay On Fri, 21 Feb 2014 20:13:33 -0600, Chris Fage cdf...@gmail.com wrote: Thanks for the assistance, everyone. For those who suggested XDS: I forgot to mention that I have tried Mosfim, which is also better than spot fitting than HKL2000. How does XDS compare to Mosflm in this regard? I am not refining the high R-factor structure with NCS options. Also, my unit cell dimensions are 41.74 A, 69.27 A, and 83.56 A, so there isn't one particularly long axis. I'm guessing the low completeness of the 1.65 angstrom dataset has to do with obstacles the processing software encountered on a sizable wedge of frames (there were swaths of in red in HKL2000). I'm not sure why this dataset in particular was less complete than the others. Thanks, Chris On Fri, Feb 21, 2014 at 6:41 PM, Chris Fage cdf...@gmail.com wrote: Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris
[ccp4bb]
Hi Vicky, I tried to change the ratio of paraffin:silicon oil to 1:2, but still the protein precipitated. So I tried to do it other way round, I kept paraffin:silicon oil ratio as 2:1 and so far it has not precipitated. Apart from that, I separately used the two oils.In silicon oil there was immediate precipitation, but none in parafin oil. Prerana
[ccp4bb]
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Prerana, is there a reason you do not try vapour diffusion instead of batch crystallisation? You can have better control about the rate of change which might affect the behaviour of the sample. Regards, Tim On 02/24/2014 05:23 PM, Prerana G. wrote: Hi Vicky, I tried to change the ratio of paraffin:silicon oil to 1:2, but still the protein precipitated. So I tried to do it other way round, I kept paraffin:silicon oil ratio as 2:1 and so far it has not precipitated. Apart from that, I separately used the two oils.In silicon oil there was immediate precipitation, but none in parafin oil. Prerana - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTC3NoUxlJ7aRr7hoRAoV1AJ9zCduZyveucwdGnxXKc+ApXS48TwCfRs5Q Aov3mv3qrfP8LGFOc68uQNE= =0/pf -END PGP SIGNATURE-
[ccp4bb] pymol multiple transparent surfaces
Dear BB-lers, I want to draw in pymol 2 objects, which are behind each other, as transparent surfaces. The surface at the back gets simply deleted. This would be correct (save some rendering time) if the object at the front was not see-thru. What wheel do I need to turn to get the surface of my object in the back drawn? Thanks a lot, Matthias -- - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk -
Re: [ccp4bb] pymol multiple transparent surfaces
Hi Matthias, you can switch to multi-layer transparency: PyMOL set transparency_mode, 1 This works for ray-tracing. In real-time (OpenGL) rendering, it currently depends on which object was enabled first. So try to toggle the back object off and on in the object menu panel. Cheers, Thomas Subject: [ccp4bb] pymol multiple transparent surfaces Date: Mon, 24 Feb 2014 22:25:39 + From: Matthias Zebisch matthias.zebi...@bbz.uni-leipzig.de Reply-To: matth...@strubi.ox.ac.uk To: CCP4BB@JISCMAIL.AC.UK Dear BB-lers, I want to draw in pymol 2 objects, which are behind each other, as transparent surfaces. The surface at the back gets simply deleted. This would be correct (save some rendering time) if the object at the front was not see-thru. What wheel do I need to turn to get the surface of my object in the back drawn? Thanks a lot, Matthias -- - Dr. Matthias Zebisch Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK Phone (+44) 1865 287549; Fax (+44) 1865 287547 Email matth...@strubi.ox.ac.uk Website http://www.strubi.ox.ac.uk - -- Thomas Holder PyMOL Developer Schrödinger, Inc.
