[ccp4bb] Postdoc position on Hsp90 Structural Biology
Postdoctoral Research Fellow in Structural Biology of Hsp90 complexes (Fixed term) Ref 143 http://www.sussex.ac.uk/aboutus/jobs/143 School of Life Sciences - Genome Damage and Stability Centre Fixed term for 3 years, full time. Salary range: starting at £31,342 and rising to £37,394 per annum. Closing date for applications: 31 May 2015 Description The School of Life Sciences is at the forefront of research in the biological sciences in the UK, coming in the top 10 in the REF 2014. A Wellcome Trust-funded postdoctoral position is available immediately in the laboratory of Professor Laurence Pearl FRS (http://www.sussex.ac.uk/lifesci/pearllab/) in the Genome Damage and Stability Centre at Sussex University, to study the structural basis for client protein recruitment and activation by the Hsp90 molecular chaperone system. We are very well equipped for all aspects of modern structural biology, with state-of-the-art laboratories for molecular biology, recombinant expression in bacterial and eukaryotic systems, biochemistry, biophysics and X-ray crystallography. Excellent synchrotron access (~2 days/month) is available through rolling beam allocation programmes at Diamond and ESRF. Applicants must have a PhD, and experience in recombinant expression and protein purification. Experience of crystallisation and X-ray crystallography and/or single particle EM, would be an advantage. Information on our previous work in this field can be found by searching PubMed (pearl [au] and hsp90). The postholder will be responsible for expression, purification, crystallization and structural analysis of protein complexes involving Hsp90, co-chaperones and client proteins that depend on Hsp90 for their biological function. Informal enquiries can be made to Professor Pearl laurence.pe...@sussex.ac.ukmailto:laurence.pe...@sussex.ac.uk. The School is committed to equality and valuing diversity, and currently holds an Athena SWAN Bronze Award. Applications are particularly welcomed from women and black and minority ethnic candidates, who are under-represented in academic posts in science and engineering at Sussex. The School of Life Sciences welcomes applications to academic posts from candidates who wish to work part-time or as job-sharers. The University offers various schemes to provide real benefits to parents, these can be found at http://www.sussex.ac.uk/humanresources/personnel/familyfriendlypolicies Laurence H. Pearl PhD FRS FMedSci Professor of Structural Biology and Head of School of Life Sciences University of Sussex, Brighton, BN1 9RH, UK Phone +44-(0)1273 876 544: PA +44-(0)1273 872 699 Live Modestly and do Serious Things .. - Dorothy Crowfoot Hodgkin
[ccp4bb] oxford cobra vs oxford 800
Dear All, Sorry for the off topic question but I would like to hear from this group experiences they have with sample cooling systems for x-ray data collection. We have had a x-stream from Rigaku since ~1999 and have been very happy with it. Unfortunately it is now obsolete since we can not get repair parts for it. We have really enjoyed the convenience of this system and were seriously contemplating upgrading to the cobra. However, I now have serious reservations due to the high cost of the service contract for this system. What have been people's experiences with either of these two systems or do you have another favorite sample cooling system we should consider. Thanks in advance, Linda Linda Olson, PhD Research Scientist II/ X-ray Facility Manager Dept Biochemistry Medical College of Wisconsin 8701 Watertown Plank Rd Milwaukee, WI 53226 phone: 414-955-8545 fax: 414-456-6510 _
[ccp4bb] Off topic - Mercury Lamp for Akta
The mercury lamp (28-4042-25) offered by GE Healthcare for Akta systems come with the housing and is priced at $1345. We have plenty of sparing housing units and are interested in just the lamp. Does anyone here have any cheaper alternatives/suggestions than buying the lamp and housing? Thanks in advance. Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 http://www.sundberglab.org/Home.html
Re: [ccp4bb] Off topic - Mercury Lamp for Akta
I wouldn't mind knowing how to source this lamp as well. But FYI, the lamps are usable for years after the FPLC gives you the dire low intensity warning. We just ignore it until the lamp goes completely dark or it's impossible to measure normal absorbances. We've used the current lamp for many, many years and it stays on all the time. ___ Roger S. Rowlett Gordon Dorothy Kline Professor 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...@colgate.edu On 5/5/2015 11:05 AM, Bonsor, Daniel wrote: The mercury lamp (28-4042-25) offered by GE Healthcare for Akta systems come with the housing and is priced at $1345. We have plenty of sparing housing units and are interested in just the lamp. Does anyone here have any cheaper alternatives/suggestions than buying the lamp and housing? Thanks in advance. Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 http://www.sundberglab.org/Home.html
Re: [ccp4bb] Off topic - Mercury Lamp for Akta
I am not sure if you have anyone electronically-minded at your disposal, however there are now LEDs available at 260 and 280nm peak wavelengths. They cost ~$40-$120 each (my data are at least 6 months old so I am not sure if they still cost the same) and they should, with a little fiddling, be able to replace the lamps - provided that you're interested in these two wavelengths :) Artem - Cosmic Cats approve of this message On Tue, May 5, 2015 at 11:05 AM, Roger Rowlett rrowl...@colgate.edu wrote: I wouldn't mind knowing how to source this lamp as well. But FYI, the lamps are usable for years after the FPLC gives you the dire low intensity warning. We just ignore it until the lamp goes completely dark or it's impossible to measure normal absorbances. We've used the current lamp for many, many years and it stays on all the time. ___ Roger S. Rowlett Gordon Dorothy Kline Professor 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...@colgate.edu On 5/5/2015 11:05 AM, Bonsor, Daniel wrote: The mercury lamp (28-4042-25) offered by GE Healthcare for Akta systems come with the housing and is priced at $1345. We have plenty of sparing housing units and are interested in just the lamp. Does anyone here have any cheaper alternatives/suggestions than buying the lamp and housing? Thanks in advance. Dan Daniel A Bonsor PhD. Sundberg Lab Institute of Human Virology University of Maryland, Baltimore 725 W Lombard Street N370 Baltimore Maryland MD 21201 Tel: (410) 706-7457 http://www.sundberglab.org/Home.html
Re: [ccp4bb] cryo condition
Along with all the excellent suggestions so far you can also try no cryoprotectant at all: If you harvest your crystal on a mesh loop and remove all the mother liquor the crystal lattice itself will act as a cryoprotectant as long as the solvent channels are smaller than 40A in diameter (they usually are). Shameless self citation: http://scripts.iucr.org/cgi-bin/paper?S0907444911031210 Best wishes, Matt. On 05/05/2015 13:16, Clemens Grimm wrote: Hi Faisal, Did you try to simply raise the Sokalan CP7 percentage? Additives like Glycerol can increase the solubility of proteins, in that case you have to counteract by increasing also the precipitant concentration. If your crystals crack, a likely reason is osmotic shock. Particularly large crystals tend to be problematic. It is in general advisable to transfer the cryo protectant onto the crystal within the mother liquor rather than fishing the crystal out with a loop. In addition, it might be necessary to prepare intermediate concentration of the cryo protectant by mixing with reservoir solution and do a stepwise increase. I had several projects with very large crystals that definitely needed at least five gradual steps over a time span of more than half an hour to extract datasets with reasonable mosaicity. Sokalan CP7 is an acrylate copolymer. Therefore, low molecular weight sodium polycralyte as a cryo protectant (e. g. Aldrich #420344) might be worth a try, possibly also in combination with some amount of good old glycerol, PEG, Trehalose, TMAO etc. Best wishes, Clemens Zitat von Faisal Tarique faisaltari...@gmail.com: Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance -- Regards Faisal School of Life Sciences JNU -- Dr. Clemens Grimm Institut für Biochemie Biozentrum der Universität Würzburg Am Hubland D-97074 Würzburg Germany e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de phone : +49 0931 31 84031 - -- Matthew Bowler Synchrotron Crystallography Group European Molecular Biology Laboratory 71 avenue des Martyrs CS 90181 F-38042 Grenoble France === Tel: +33 (0) 4.76.20.76.37 Fax: +33 (0) 4.76.20.71.99 http://www.embl.fr/ ===
Re: [ccp4bb] Tev cleavage
You could try adding TEV to your protein just prior to crystallisation. I produced 2 equivalent structures this way; one with the tag clearly visible packing between molecules in an I centred space group and a 2nd (with TEV added) in P21 where the tag was no longer visible (the packing suggested it would not be accommodated and therefore cleaved). I don’t think many people try detagging their protein in conditions as extreme as those in a crystallisation trial but in the end that’s where the protein needs to be happy. Yes, there are lots of caveats with this technique, not least sub optimal cleavage conditions, but it’s a very simple experiment to do. Dave From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Giulliana Rangel Sent: 02 May 2015 18:57 To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Tev cleavage Dear all, I'd like some help about my protein cause I've a lot of problems in cleavage moment. In this step after aproximadately 30 minutes (37C) occur precipitation almost 50% . I tried control it: - Protein diluition (no results) - Cleavage 4C ( no cleavage) -Modifying buffers: add 10% glycerol and 5% glucose (no crystallization) - Add salt (1M - no results) - Add serial tev (500ul in the first time and more 500ul in second time- 37C) total precipitation - Crystallization with 7 histag ( poor crystallization, no diffraction) Now I need to produce this protein with semet that became the protein more hidrofobic, probably. So, If anyone could help me... Thanks in advance Giulliana Rangel AstraZeneca UK Limited is a company incorporated in England and Wales with registered number: 03674842 and a registered office at 2 Kingdom Street, London, W2 6BD. Confidentiality Notice: This message is private and may contain confidential, proprietary and legally privileged information. If you have received this message in error, please notify us and remove it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorised use or disclosure of the contents of this message is not permitted and may be unlawful. Disclaimer: Email messages may be subject to delays, interception, non-delivery and unauthorised alterations. Therefore, information expressed in this message is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by an authorised representative independent of this message. No contractual relationship is created by this message by any person unless specifically indicated by agreement in writing other than email. Monitoring: AstraZeneca UK Limited may monitor email traffic data and content for the purposes of the prevention and detection of crime, ensuring the security of our computer systems and checking compliance with our Code of Conduct and policies.
Re: [ccp4bb] Question for those who use AutoSharp via CCP4i on MacOSX Yosemite
I’ll add to this that you should also set the relevant path in launchd.conf as well to access other external programs (SHELXC/D/E) when opening ccp4i via the Finder/Spotlight. setenv PATH /where/ever/shelx Cheers, Aaron -- Dr. Aaron Finke Postdoctoral Fellow Swiss Light Source WSLA/217 CH-5232 Villigen-PSI phone: +41 56 310 5652 e-mail: aaron.fi...@psi.chmailto:aaron.fi...@psi.ch On May 5, 2015, at 11:00, Finke Aaron (PSI) aaron.fi...@psi.chmailto:aaron.fi...@psi.ch wrote: Dear Mark, Indeed there is a way. You have to set the environment variable so that all Mac apps can access it. This is most easily done via launchd. In the Terminal, type ‘sudo vi /etc/launchd.conf’ and add the following text (the file will likely be blank): setenv SHARP_home /where/ever/SHARP/ Save, and reboot. Now when you access ccp4i in a Finder window or Spotlight, autoSHARP should be accessible. More info can be found here: http://stackoverflow.com/questions/135688/setting-environment-variables-in-os-x/3756686#3756686 I hope this works for you. (It worked for me.) Cheers, Aaron -- Dr. Aaron Finke Postdoctoral Fellow Swiss Light Source WSLA/217 CH-5232 Villigen-PSI phone: +41 56 310 5652 e-mail: aaron.fi...@psi.chmailto:aaron.fi...@psi.ch On May 5, 2015, at 1:17, Mark J van Raaij mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote: Dear All, when starting CCP4i via the icon, AutoSharp remains greyed out, suggesting it is not installed. However, when I start CCP4i in an XQuartz window (i.e. “ccp4i ”), AutoSharp can be run. I guess this may be because in first way my “.profile” file is not read, any ideas how to fix this? Greetings, Mark J van Raaij mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es http://www.cnb.csic.es/~mjvanraaij
Re: [ccp4bb] Cryo protection
Cryo On 5 May 2015 13:51, faisaltari...@gmail.com wrote: Thank you everyone..I have got some hints from this discussion and will definitely try some of them to check its efficacy for my case...Thanx again for your valuable suggestions.. On 5 May 2015 13:48, Anthony Savill t...@moleculardimensions.com wrote: Dear colleagues, Having followed the CCP4 BB discussion on cryoprotection I thought you might be interested to know that with the help of work from Enrico Stura's lab Molecular Dimension have introduced two kits now to take some of the trial and error out of finding an effective cryoprotectant. http://www.moleculardimensions.com/shopdisplayproducts.asp?id=205cat=CryoKits Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography. Crystal Growth Design, http://pubs.acs.org/doi/full/10.1021/cg301531f Tony Savill Molecular Dimensions Ltd. Unit 6 Goodwin Business Park Willie Snaith Road Newmarket, Suffolk. UK CB8 7SQ Tel: +44 1638 561051 Fax: +44 1638 660674 Registered Office Salisbury House Station Road Cambridge CB1 2LA Registered in England and Wales: Registration number 1794026
Re: [ccp4bb] Question for those who use AutoSharp via CCP4i on MacOSX Yosemite
Dear Mark, Indeed there is a way. You have to set the environment variable so that all Mac apps can access it. This is most easily done via launchd. In the Terminal, type ‘sudo vi /etc/launchd.conf’ and add the following text (the file will likely be blank): setenv SHARP_home /where/ever/SHARP/ Save, and reboot. Now when you access ccp4i in a Finder window or Spotlight, autoSHARP should be accessible. More info can be found here: http://stackoverflow.com/questions/135688/setting-environment-variables-in-os-x/3756686#3756686 I hope this works for you. (It worked for me.) Cheers, Aaron -- Dr. Aaron Finke Postdoctoral Fellow Swiss Light Source WSLA/217 CH-5232 Villigen-PSI phone: +41 56 310 5652 e-mail: aaron.fi...@psi.chmailto:aaron.fi...@psi.ch On May 5, 2015, at 1:17, Mark J van Raaij mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es wrote: Dear All, when starting CCP4i via the icon, AutoSharp remains greyed out, suggesting it is not installed. However, when I start CCP4i in an XQuartz window (i.e. “ccp4i ”), AutoSharp can be run. I guess this may be because in first way my “.profile” file is not read, any ideas how to fix this? Greetings, Mark J van Raaij mjvanra...@cnb.csic.esmailto:mjvanra...@cnb.csic.es http://www.cnb.csic.es/~mjvanraaij
[ccp4bb] Formulatrix NT8
Hi, I would like to come into contact with users of the Formulatrix NT8, especially if they have the LCP option. If you have one, please contact me off-list. Best regards Derek Derek Logan tel: +46 46 222 1443 Associate Professor mob: +46 76 8585 707 Dept. of Biochemistry and Structural Biology www.cmps.lu.sehttp://www.cmps.lu.se Centre for Molecular Protein Sciencewww.maxlab.lu.se/crystal Lund University, Box 124, 221 00 Lund, Sweden
Re: [ccp4bb] cryo condition
Hi Faisal, Did you try to simply raise the Sokalan CP7 percentage? Additives like Glycerol can increase the solubility of proteins, in that case you have to counteract by increasing also the precipitant concentration. If your crystals crack, a likely reason is osmotic shock. Particularly large crystals tend to be problematic. It is in general advisable to transfer the cryo protectant onto the crystal within the mother liquor rather than fishing the crystal out with a loop. In addition, it might be necessary to prepare intermediate concentration of the cryo protectant by mixing with reservoir solution and do a stepwise increase. I had several projects with very large crystals that definitely needed at least five gradual steps over a time span of more than half an hour to extract datasets with reasonable mosaicity. Sokalan CP7 is an acrylate copolymer. Therefore, low molecular weight sodium polycralyte as a cryo protectant (e. g. Aldrich #420344) might be worth a try, possibly also in combination with some amount of good old glycerol, PEG, Trehalose, TMAO etc. Best wishes, Clemens Zitat von Faisal Tarique faisaltari...@gmail.com: Hello everyone Can anybody suggest me a cryo condition for a crystal obtained in MIDAS screen of Molecular Dimension: G1 0.1MTris8.0G10.1Mpotassium chloride25% v/vSOKALAN®CP7 0.1MHEPES7.0 G20.3Mammonium formate20% v/vSOKALAN® CP 5 0.1MHEPES7.0 Crystals are in beautiful cuboid shaped but all sorts of PEG combinations and Glycerol formulation failed to prevent it from cracking and dissolving. Has anybody faced a similar situation as mentioned above and what precaution was taken to prevent it from cracking or dissolving. Your suggestions will be of immense help Thanks in advance -- Regards Faisal School of Life Sciences JNU -- Dr. Clemens Grimm Institut für Biochemie Biozentrum der Universität Würzburg Am Hubland D-97074 Würzburg Germany e-mail: clemens.gr...@biozentrum.uni-wuerzburg.de phone : +49 0931 31 84031 -
