[ccp4bb] possible Refmac_5.6.0117 bug
Happen to come across this: Failed to rerun previous refmac job, which had been run in version 5.5.0109. The problem was gone after changing the LIBIN filename (36 characters) to a shorter one. Minglei Zhao
Re: [ccp4bb] Help! weird thing
Dear Zhihong, Refinement stuck at 55% Rfree (which is essentially random), means that you do not have found the correct MR solution. For me the prime suspect is the space group. In my experience pseudo translation or any almost crystallographic NCS will easily confuse automatic space group determination programs like pointless and it is often trickey to find out which symmetry is crystallographic and which is non-crystallographic. Since P21 is fairly low symmetry, I would reprocess your data in P1 and just naively run all your favorite molecular replacement programs (phaser, molrep, epmr, phenix, etc.) in P1. I would try them all since there are subtle difference between these programs and one may succeed where the others fail. Once you have a solution which refines (Free Rfactor drops below say 40% in P1) you can try to figure out what the true, higher symmetry space group may be. Your true space group may even be P1, with pseudo P21 symmetry! Good luck! Herman From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of xiaoyazi2008 Sent: Thursday, March 15, 2012 6:12 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Help! weird thing Dear all, Thank you very much for all the great suggestions on my case. Yes, I run the latest version of Phaser in Phenix. The analysis showed that there is one non-origin distinct peak more than 15 angstroms from the origin. 44.1% origin:FRAC 0.000 0.042 0.500 (ORTH -15.72.8 103.5) I managed to find four copies with the latest Phaser. After 50 cycles of rigid body refinement and 50 cycles of jelly body refinement, Rfree/R goes around 55/52. It is really hard for me to do model building at this point, because there is pretty much no new density. Compare to model building and refinement with normal dataset (no pseudo translation NCS), are there any special tricks or tips for structure determination from dataset with pseudo translation? Thanks again! Have a nice evening or morning or afternoon! Zhihong On Mar 12, 2012, at 10:16 AM, Randy Read wrote: Airlie points out that what I said about the ccp4i interface wasn't correct! In order to keep the ccp4i interface in synch with the version of Phaser, we've started distributing the ccp4i files with the source code. The ones on our website are for an older version of Phaser, but the latest ones will come with the Phenix download that gives you the latest executable. Apologies to anyone who was quick enough to download and install the wrong ccp4i files already! Best wishes, Randy Read On 12 Mar 2012, at 16:47, Randy Read wrote: Yes, the current version of Phaser will do the same test that xtriage carries out, and if it finds a sufficiently high non-origin Patterson peak, it will automatically characterise the translational NCS and use this for molecular replacement. This is working pretty well in our tests. In the near future you will be able to get the current version of Phaser as part of the upcoming CCP4 release, but at the moment the easiest way to get it is to download a recent version of Phenix. You should be able to run that through ccp4i by downloading and installing the updated GUI files from our website (and getting ccp4i to interpret the command phaser as phenix.phaser). Best wishes, Randy Read On 12 Mar 2012, at 16:06, Anastassis Perrakis wrote: Hi - I agree with Garib that its likely a pseudo-translation issue. I also agree with that the advice he gives is correct, but ... ... since I am evidently less smart to follow all these steps, I like to use phenix.xtriage that will tell me if there is pseudo-translation or not, and will give a p-value for that being significant. Its at the end of the text output. I am not sure if Phaser deals these days with pseudo-translation - I guess Randy can tell us. If not, there is a very simple trick to make Phaser work with pseudo-translation, but since I threw the ball to Randy's court and he told me the trick a few years ago, I will let him explain only if needed ;-) Best, Tassos On Mar 11, 2012, at 12:55, Garib N Murshudov wrote: Hi Could you please check:
[ccp4bb] MolRep error
Dear All, I am trying to run MolRep with 'input fixed model' option and i am getting error message: At line 1260 of file /usr/local/xtal/ccp4-6.2.0/src/molrep_/molrep.f Fortran runtime error: End of record I am using: CCP4 6.2: MOLREP(ccp4) version 11.0.02 : 07/02/11 I found the updated version of the molrep binary for Windows system but could not see anything for linux. I'll be grateful for your help. Justyna -- Dr Justyna Aleksandra Wojdyla Department of Biology University of York Heslington, York, YO10 5DD United Kingdom Tel: +44 (0)1904 328818 Email: justyna.wojd...@york.ac.uk EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm
Re: [ccp4bb] MolRep error
you can try a version from this site: http://www.ysbl.york.ac.uk/~alexei/molrep.html If it does not work please let me know regards Garib On 15 Mar 2012, at 12:45, Justyna Wojdyla jw...@york.ac.uk wrote: Dear All, I am trying to run MolRep with 'input fixed model' option and i am getting error message: At line 1260 of file /usr/local/xtal/ccp4-6.2.0/src/molrep_/molrep.f Fortran runtime error: End of record I am using: CCP4 6.2: MOLREP(ccp4) version 11.0.02 : 07/02/11 I found the updated version of the molrep binary for Windows system but could not see anything for linux. I'll be grateful for your help. Justyna -- Dr Justyna Aleksandra Wojdyla Department of Biology University of York Heslington, York, YO10 5DD United Kingdom Tel: +44 (0)1904 328818 Email: justyna.wojd...@york.ac.uk EMAIL DISCLAIMER http://www.york.ac.uk/docs/disclaimer/email.htm Garib N Murshudov Structural Studies Division MRC Laboratory of Molecular Biology Hills Road Cambridge CB2 0QH UK Email: ga...@mrc-lmb.cam.ac.uk Web http://www.mrc-lmb.cam.ac.uk
[ccp4bb] Lego_auto_mainchain in COOT?
Dear all, Is there any similar function in COOT as lego_auto_mainchain command in O program? This command is quite useful to build a low resolution model (say, 4A). Cheers, Xiuwen
Re: [ccp4bb] Lego_auto_mainchain in COOT?
On 15/03/12 14:14, xiuwen zhang wrote: Dear all, Is there any similar function in COOT as lego_auto_mainchain command in O program? This command is quite useful to build a low resolution model (say, 4A). I am not familiar with lego_auto_mainchain but there are 2 loop fitting tools in Coot C alpha - Mainchain http://lmb.bioch.ox.ac.uk/coot/doc/coot.html#C_002dalpha-_002d_003e-Mainchain http://lmb.bioch.ox.ac.uk/coot/doc/coot.html#Building-Links-and-Loops DB Loop: (No good documentation) http://lmb.bioch.ox.ac.uk/coot/doc/coot/protein_002ddb_002dloops.html#protein_002ddb_002dloops Extensions - Modelling - DB Loop...
[ccp4bb] protein stain, B-PER
Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - B-PER Bacterial Protein Extraction Reagents are designed to extract soluble protein from bacterial cells without harsh chemicals or mechanical procedures like sonication), but would like to try a variety of similar things if possible. Any advice from the community out there? Anybody know what goes into B-PER or similar things (I know there's some Dnase and lysosyme in there – but which detergents are compatible with Ni, GST, how much do you need etc)?? 2. Staining SDS gels. There are various concerns from lab members about safety re methanol in stains, microwaving stains etc etc. Instant Blue claims to have none of these problems. Quote: Protein gel staining takes around 15 minutes without the need to wash, fix, microwave or destain. But again, I'd like to try things to see if they work for us (before spending cash - yes, I am spending averse…!). Anybody any suggestions for quick, non-fix, non-methanol, non-microwave, non–destain protein gel stains? Have tried home made colloidal coomassie but our protocol still requires fixes and washes that made it not really worth while. Happy to collate thoughts on replies offline and post summary. Many thanks Ed T.A.Edwards Ph.D. Deputy Director Astbury Centre for Structural Molecular Biology Lecturer in Biochemistry Garstang 8.53d University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- No one should approach the temple of science with the soul of a money changer. ~Thomas Browne
Re: [ccp4bb] protein stain, B-PER
Switch ethanol in the stain. Also, there was talk a while ago about making your own instant blue stains--it works like bradford reagent, I think. Perhaps look back at previous postings? JPK On Thu, Mar 15, 2012 at 9:24 AM, Thomas Edwards t.a.edwa...@leeds.ac.ukwrote: Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - B-PER Bacterial Protein Extraction Reagents are designed to extract soluble protein from bacterial cells without harsh chemicals or mechanical procedures like sonication), but would like to try a variety of similar things if possible. Any advice from the community out there? Anybody know what goes into B-PER or similar things (I know there's some Dnase and lysosyme in there – but which detergents are compatible with Ni, GST, how much do you need etc)?? 2. Staining SDS gels. There are various concerns from lab members about safety re methanol in stains, microwaving stains etc etc. Instant Blue claims to have none of these problems. Quote: Protein gel staining takes around 15 minutes without the need to wash, fix, microwave or destain. But again, I'd like to try things to see if they work for us (before spending cash - yes, I am spending averse…!). Anybody any suggestions for quick, non-fix, non-methanol, non-microwave, non–destain protein gel stains? Have tried home made colloidal coomassie but our protocol still requires fixes and washes that made it not really worth while. Happy to collate thoughts on replies offline and post summary. Many thanks Ed T.A.Edwards Ph.D. Deputy Director Astbury Centre for Structural Molecular Biology Lecturer in Biochemistry Garstang 8.53d University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- No one should approach the temple of science with the soul of a money changer. ~Thomas Browne -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] protein stain, B-PER
Hi Ed, A few years back there has been a thread on copper staining that leads to negative staining, but works well, is very fast and simple. - Rinse gel 10-30s in H2O - incubate in 300 mM CuCl2. The gel stains in 3-5 min: background becomes opaque, the protein bands remain clear. Sensibility is as good (slightly better) than Coomassie. The gel can be stored in H2O (but proteins diffuse after a while), destained for other staining (Coomassie, silver...), by incubation in 50 mM EDTA 5-10 min. CuCl2 and EDTA solutions can be re-used. Discard Cu in appropriate waste. From Lee et al, 1987, Anal. Biochem 166, 308-312. Cécile Le 15/03/12 15:24, Thomas Edwards a écrit : Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - B-PER Bacterial Protein Extraction Reagents are designed to extract soluble protein from bacterial cells without harsh chemicals or mechanical procedures like sonication), but would like to try a variety of similar things if possible. Any advice from the community out there? Anybody know what goes into B-PER or similar things (I know there's some Dnase and lysosyme in there – but which detergents are compatible with Ni, GST, how much do you need etc)?? 2. Staining SDS gels. There are various concerns from lab members about safety re methanol in stains, microwaving stains etc etc. Instant Blue claims to have none of these problems. Quote: Protein gel staining takes around 15 minutes without the need to wash, fix, microwave or destain. But again, I'd like to try things to see if they work for us (before spending cash - yes, I am spending averse…!). Anybody any suggestions for quick, non-fix, non-methanol, non-microwave, non–destain protein gel stains? Have tried home made colloidal coomassie but our protocol still requires fixes and washes that made it not really worth while. Happy to collate thoughts on replies offline and post summary. Many thanks Ed T.A.Edwards Ph.D. Deputy Director Astbury Centre for Structural Molecular Biology Lecturer in Biochemistry Garstang 8.53d University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- No one should approach the temple of science with the soul of a money changer. ~Thomas Browne -- Cécile Breyton Institut de Biologie Structurale UMR 5075 CNRS/CEA/UJF 41, rue Jules Horowitz 38027 Grenoble cedex 1 France --- Tel: +33 (0)4 38 78 30 37 Fax: +33 (0)4 38 78 54 94 Courriel : cecile.brey...@ibs.fr http://www.ibs.fr/groups/membrane-and-pathogens-group/ssimpa/article/ssimpas-1109
Re: [ccp4bb] protein stain, B-PER
By mechanical disruption you mean sonication only or have you tried the French press? Assuming that you use sonication, and assuming that you follow a fairly standard protocol (e.g. something like 10sec pulse/20sec pause on ice for 3 minutes total), it may be heat not ultrasound that gets to it. Temperature in the sonicated sample may go pretty high under regular sonication protocol - we have the temperature probe and it easily goes over room temperature. I may certainly be wrong on both assumptions. And French press would suffer from the same heating problem - some pre-cool the whole thing in the cold room, but I don't know how much it helps. Cheers, Ed. On Thu, 2012-03-15 at 14:24 +, Thomas Edwards wrote: Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - B-PER Bacterial Protein Extraction Reagents are designed to extract soluble protein from bacterial cells without harsh chemicals or mechanical procedures like sonication), but would like to try a variety of similar things if possible. Any advice from the community out there? Anybody know what goes into B-PER or similar things (I know there's some Dnase and lysosyme in there – but which detergents are compatible with Ni, GST, how much do you need etc)?? 2. Staining SDS gels. There are various concerns from lab members about safety re methanol in stains, microwaving stains etc etc. Instant Blue claims to have none of these problems. Quote: Protein gel staining takes around 15 minutes without the need to wash, fix, microwave or destain. But again, I'd like to try things to see if they work for us (before spending cash - yes, I am spending averse…!). Anybody any suggestions for quick, non-fix, non-methanol, non-microwave, non–destain protein gel stains? Have tried home made colloidal coomassie but our protocol still requires fixes and washes that made it not really worth while. Happy to collate thoughts on replies offline and post summary. Many thanks Ed T.A.Edwards Ph.D. Deputy Director Astbury Centre for Structural Molecular Biology Lecturer in Biochemistry Garstang 8.53d University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- No one should approach the temple of science with the soul of a money changer. ~Thomas Browne -- Hurry up before we all come back to our senses! Julian, King of Lemurs
Re: [ccp4bb] protein stain, B-PER
Hey, that was my post! NB this cannot be used for native gels, I don't think. Jacob On Thu, Mar 15, 2012 at 9:47 AM, Cécile Breyton cecile.brey...@ibs.frwrote: Hi Ed, A few years back there has been a thread on copper staining that leads to negative staining, but works well, is very fast and simple. - Rinse gel 10-30s in H2O - incubate in 300 mM CuCl2. The gel stains in 3-5 min: background becomes opaque, the protein bands remain clear. Sensibility is as good (slightly better) than Coomassie. The gel can be stored in H2O (but proteins diffuse after a while), destained for other staining (Coomassie, silver...), by incubation in 50 mM EDTA 5-10 min. CuCl2 and EDTA solutions can be re-used. Discard Cu in appropriate waste. From Lee et al, 1987, Anal. Biochem 166, 308-312. Cécile Le 15/03/12 15:24, Thomas Edwards a écrit : Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - B-PER Bacterial Protein Extraction Reagents are designed to extract soluble protein from bacterial cells without harsh chemicals or mechanical procedures like sonication), but would like to try a variety of similar things if possible. Any advice from the community out there? Anybody know what goes into B-PER or similar things (I know there's some Dnase and lysosyme in there – but which detergents are compatible with Ni, GST, how much do you need etc)?? 2. Staining SDS gels. There are various concerns from lab members about safety re methanol in stains, microwaving stains etc etc. Instant Blue claims to have none of these problems. Quote: Protein gel staining takes around 15 minutes without the need to wash, fix, microwave or destain. But again, I'd like to try things to see if they work for us (before spending cash - yes, I am spending averse…!). Anybody any suggestions for quick, non-fix, non-methanol, non-microwave, non–destain protein gel stains? Have tried home made colloidal coomassie but our protocol still requires fixes and washes that made it not really worth while. Happy to collate thoughts on replies offline and post summary. Many thanks Ed T.A.Edwards Ph.D. Deputy Director Astbury Centre for Structural Molecular Biology Lecturer in Biochemistry Garstang 8.53d University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/**staff/tae/http://www.bmb.leeds.ac.uk/staff/tae/ -- No one should approach the temple of science with the soul of a money changer. ~Thomas Browne -- Cécile Breyton Institut de Biologie Structurale UMR 5075 CNRS/CEA/UJF 41, rue Jules Horowitz 38027 Grenoble cedex 1 France --- Tel: +33 (0)4 38 78 30 37 Fax: +33 (0)4 38 78 54 94 Courriel : cecile.brey...@ibs.fr http://www.ibs.fr/groups/**membrane-and-pathogens-group/** ssimpa/article/ssimpas-1109http://www.ibs.fr/groups/membrane-and-pathogens-group/ssimpa/article/ssimpas-1109 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] protein stain, B-PER
Hi all, concerning the staining, we get good results with a self-made instant blue. First staining is achieved within 30min, full staining after maximal 4 hours I would say. You don't need to destain (still possible with H2O) since the background is not stained. Sensitivity is better than standard CBB-G250/Acetic Acid staining (not as good as silver stain though). Staining recipe: 10% EtOH 2% phosphoric acid 5% (w/v) AlSO4 0.02% CBB-G250 The phosphoric acid might be problematic in this mixture, but that would be my only concern. At least you don't have to heat it plus you have higher sensitivity. Check this paper for more information: Fast and sensitive colloidal coomassie G-250 staining for proteins in polyacrylamide gels. Dyballa, N. Metzger, S. PMID: 19684561 Best
[ccp4bb] Reminder: BCA/CCP4 Summer School 2012
This is a reminder that applications are now open for the BCA/CCP4 Summer School in Protein Crystallography to be held at Diamond (UK) from Tuesday 28th August to Sunday 2nd September 2012. Applications close 1st May 2012. The course is suitable for PhD students in macromolecular crystallography who started in or before October 2011, and postdocs new to macromolecular crystallography. Please note that references from supervisors are also due by 1st May 2012, so don't leave your application until the last minute! For more information see: http://www.diamond.ac.uk/Home/Events/BCA-Summer-School-2012.html Application form: http://www.diamond.ac.uk/Home/Events/BCA-Summer-School-2012/Application-Page.html
[ccp4bb] Experimental Phasing with Pseudo-symmetry in an unusual setting P 21 2 21
Dear colleagues do you have any professional thoughts on my case here? Available SeMet data to 3.6 Å (where only the redundant Se-Edge peak to can be used), a high resolution native data to 2.6 Å, a Zinc-soak, collected at the Zinc-Edge to 3.0 Å. Considerable Patterson peak indicates pseudo translational symmetry, as of 42% of the origin peak. The fractional vector (x,y,z) is 0, 0.22, 0.5. The unit cell at 65 Å, 104 Å, 130 Å (all angles 90 deg) is expected to contain 2 copies at 51% solvent content. Best solutions for the substructure with 6 of the expected 12 Selenium sites found in P 21 2 21 (sometimes called 2018). Low CC of ~0.4 and problematic to refine and or find new sites after density modification. Molecular Replacement (with and without assisted SAD) did not work, i.e. R-factors 50% and CC 0.4. With the very best slightly manually trimmed and refined solution I see R/Rfree 0.47/0.51. Programs in use are shelx_cde, autoSHARP, ARP/wARP, crank, oasis, rantan, phaser, mlphare, as well as phenix.autosol, phase_and_build, refine, sculptor, enseml, automr. 1) Can I reassure myself of the right space group? (tried visual absences inspection in HKLVIEW and pointless, phenix.xtriage) 2) What is the best phasing program at these resolutions in your experience? Thanks in advance for your feedback! Harm --- Harm Otten, PhD Office C316 Biophysical Chemistry Group Department of Chemistry Universitetsparken 5 2100 Copenhagen Denmark # +45 35 32 02 86 fax +45 35 32 03 22 email h...@chem.ku.dk Please consider the environment before printing this email.
[ccp4bb] Differentiate salt and protein crystals
Hi all. I set up some trays of membrane protein remotely in cubic phase. I don't have ready access to them so I can't shoot/pick the crystals. Under polarising lights, some crystals appears coloured across many conditions, making me think these are salt. Is there some knowledge of inorganic chemistry that be relied to prioritise some for reproductions at my lab? Can detergents crystallise and produce colours under polarising light? Thank you. Theresa
Re: [ccp4bb] Differentiate salt and protein crystals
Hi Theresa, Your observation of colored crystals under polarising lights seems odd to me. I assume your protein should be colorless and under normal light these crystals are colorless? Are these trays set up in glass plate or plastic plates? If you are willing to upload some pictures, I might be able to tell you whether they are salt crystals or not. Regards Jingquan On 15 March 2012 15:43, Theresa H. Hsu theresah...@live.com wrote: Hi all. I set up some trays of membrane protein remotely in cubic phase. I don't have ready access to them so I can't shoot/pick the crystals. Under polarising lights, some crystals appears coloured across many conditions, making me think these are salt. Is there some knowledge of inorganic chemistry that be relied to prioritise some for reproductions at my lab? Can detergents crystallise and produce colours under polarising light? Thank you. Theresa
Re: [ccp4bb] Differentiate salt and protein crystals
The best reproduction I can suggest would be to setup one or two LCP experiments exchanging the protein for its buffer. If you get crystals, you know for sure they're not protein. Cheers, Jon 2012/3/15 Theresa H. Hsu theresah...@live.com Hi all. I set up some trays of membrane protein remotely in cubic phase. I don't have ready access to them so I can't shoot/pick the crystals. Under polarising lights, some crystals appears coloured across many conditions, making me think these are salt. Is there some knowledge of inorganic chemistry that be relied to prioritise some for reproductions at my lab? Can detergents crystallise and produce colours under polarising light? Thank you. Theresa -- Dr. Jon Agirre Postdoctoral Scientist - Protein and Virus X-ray Crystallography Group Biophysics Unit (CSIC-UPV/EHU) +0034946013357
Re: [ccp4bb] recombinant enterokinase
Dear Elias, we routinely produce a C-terminally His6-tagged catalytic domain of bovine enterokinase in E. coli. The protease is expressed into inclusion bodies and subsequently folded in vitro. Considering the high activity of our enterokinase construct, one refolding batch allows us to cleave several grams of target proteins. If you're interested in our expression system, just send me an email. best wishes, Wolfgang Skala -- Structural Biology Group / Department of Molecular Biology University of Salzburg Billrothstraße 11 5020 Salzburg Austria Email: wolfgang.sk...@sbg.ac.at Phone: +43 662 8044 7278 http://www.uni-salzburg.at/xray
Re: [ccp4bb] Differentiate salt and protein crystals
On Thu, Mar 15, 2012 at 11:14 AM, Jon Agirre jon.agi...@gmail.com wrote: The best reproduction I can suggest would be to setup one or two LCP experiments exchanging the protein for its buffer. If you get crystals, you know for sure they're not protein. It should be pointed out that the converse is not true: if you don't get crystals from buffer-only crystallizations, it doesn't mean that your putative protein crystals are really protein. Jacob Cheers, Jon 2012/3/15 Theresa H. Hsu theresah...@live.com Hi all. I set up some trays of membrane protein remotely in cubic phase. I don't have ready access to them so I can't shoot/pick the crystals. Under polarising lights, some crystals appears coloured across many conditions, making me think these are salt. Is there some knowledge of inorganic chemistry that be relied to prioritise some for reproductions at my lab? Can detergents crystallise and produce colours under polarising light? Thank you. Theresa -- Dr. Jon Agirre Postdoctoral Scientist - Protein and Virus X-ray Crystallography Group Biophysics Unit (CSIC-UPV/EHU) +0034946013357 -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] postdoctoral position in structural biology and epigenetics
A post-doctoral position is available for highly motivated candidates to join a research group interested in investigating molecular mechanisms underlying epigenetic regulation and inheritance, and the broad area of chromatin biology. Projects will involve structure determination of macromolecular complexes by X-ray crystallography and/or NMR spectroscopy, and related biochemical characterizations. Applicants should hold a PhD degree and have significant experience in molecular cloning, protein production and X-ray crystallography. Additional background in NMR spectroscopy and enzymatic assay is considered a plus but not essential. The expected starting date is September 1st, 2012 or sooner. Interested applicants should send curriculum vitae, a summary of past research experience and accomplishments, and contact information of two or three references to Jikui Song, Ph.D. Department of Biochemistry, University of California, Riverside, CA 92521 (Email: songlab...@gmail.edu). As one of the major research univerisities in California, UCR houses well equipped structural biology facilities. Riverside is located near to big cities such as Los Angeles, San Diego and Las Vegas. Such an environment allows one to have a good balance of life and science here. More information can be found in http://biochemistry.ucr.edu/faculty/song/song.html. Jikui Song, PhD Assistant Professor Department of Biochemistry University of California, Riverside
[ccp4bb] postdoctoral position in structural biology and epigenetics
A post-doctoral position is available for highly motivated candidates to join aresearch group interested in investigating molecular mechanisms underlyingepigenetic regulation and inheritance, and the broad area of chromatin biology.Projects will involve structure determination of macromolecular complexes byX-ray crystallography and/or NMR spectroscopy, and related biochemicalcharacterizations. Applicants should hold a PhD degree and have significantexperience in molecular cloning, protein production and X-ray crystallography.Additional background in NMR spectroscopy and enzymatic assay is considered aplus but not essential. The expected starting date is September 1st,2012 or sooner. Interested applicants should send curriculum vitae, a summaryof past research experience and accomplishments, and contact information of twoor three references to Jikui Song, Ph.D. Department of Biochemistry, Universityof California, Riverside, CA 92521 (Email: songlab...@gmail.edu). As one of the major research univerisities in California, UCR houses well-equipped X-ray and NMR facilities. Riverside is located near to big cities such as Los Angeles, San Diego and Las Vegas. Such an environment allows one to have a good balance of life and science here. More information can be found in http://biochemistry.ucr.edu/faculty/song/song.html. Jikui Song, PhD Assistant Professor Department of Biochemistry University of California,Riverside
Re: [ccp4bb] protein stain, B-PER
1. We like Bugbuster. You can get it at 10X concentration so it is fairly economical. We do add lysozyme and DNase as well. 2. We use the NuPage gels and SimplyBlue SafeStain and it works very well. We can run a gel in 20-30 min and have it stained and destained a hour later using the microwave to heat the solutions. There is no methanol in the procedure and gel runs at neutrality. Doug Douglas H. Ohlendorf Phone: 612-624-8436 Professor FAX: 612-624-5121 Dept. of Biochemistry, Molecular Biology Biophysics Twin Cities Campus, University of Minnesota Lab web site: http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Thomas Edwards Sent: Thursday, March 15, 2012 9:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein stain, B-PER Dear BB, Apologies for being mildly off topic. Maybe. 1. We are trying to express (in E. coli) a protein which appears to be quite sensitive to mechanical disruption. We have ordered some B-PER (Pierce - B-PER Bacterial Protein Extraction Reagents are designed to extract soluble protein from bacterial cells without harsh chemicals or mechanical procedures like sonication), but would like to try a variety of similar things if possible. Any advice from the community out there? Anybody know what goes into B-PER or similar things (I know there's some Dnase and lysosyme in there - but which detergents are compatible with Ni, GST, how much do you need etc)?? 2. Staining SDS gels. There are various concerns from lab members about safety re methanol in stains, microwaving stains etc etc. Instant Blue claims to have none of these problems. Quote: Protein gel staining takes around 15 minutes without the need to wash, fix, microwave or destain. But again, I'd like to try things to see if they work for us (before spending cash - yes, I am spending averse.!). Anybody any suggestions for quick, non-fix, non-methanol, non-microwave, non-destain protein gel stains? Have tried home made colloidal coomassie but our protocol still requires fixes and washes that made it not really worth while. Happy to collate thoughts on replies offline and post summary. Many thanks Ed T.A.Edwards Ph.D. Deputy Director Astbury Centre for Structural Molecular Biology Lecturer in Biochemistry Garstang 8.53d University of Leeds, Leeds, LS2 9JT Telephone: 0113 343 3031 http://www.bmb.leeds.ac.uk/staff/tae/ -- No one should approach the temple of science with the soul of a money changer. ~Thomas Browne
[ccp4bb] 4Fe-4S
Hi all, Has any of you encountered a protein with 4Fe-4S cluster; with three of the Fe coordinated to the cysteines of the protein, leaving one Fe atom free for electron transfer? The closest I got is aconitase; however, the iron sulfur cluster of the enzyme is not used for electron transfer. Thanks! Allan -- Allan Pang PhD Student G35 Joseph Priestley Building Queen Mary University of London London E1 4NS Phone number: 02078828480 Twitter: @xerophytes
[ccp4bb] Post-doctoral Position Available in Structural Biology and Epigenetics
Sorry for posting again, previous message contains a typo. A post-doctoral position is available for highly motivated candidates to join a research group interested in investigating molecular mechanisms underlying epigenetic regulation and inheritance, and the broad area of chromatin biology. Projects will involve structure determination of macromolecular complexes by X-ray crystallography and/or NMR spectroscopy, and related biochemical characterizations. Applicants should hold a PhD degree and have significant experience in molecular cloning, protein production and X-ray crystallography. Additional background in NMR spectroscopy and enzymatic assay is considered a plus but not essential. The expected starting date is September 1st,2012 or sooner. Interested applicants should send curriculum vitae, a summary of past research experience and accomplishments, and contact information of two or three references to Jikui Song, Ph.D. Department of Biochemistry, University of California, Riverside, CA 92521 (Email: songlab...@gmail.com). As one of the major research universities in California, UCR houses well-equipped X-ray and NMR facilities. Riverside is located near to big cities such as Los Angeles, San Diego and Las Vegas. Such an environment allows one to have a good balance of life and science here. More information can be found in http://biochemistry.ucr.edu/faculty/song/song.html. --- Jikui Song, PhD Department of Biochemistry University of California, Riverside
