Re: [ccp4bb] suggestions for cryoprotectant
Hi Firdous, For salt conditions try malonate: Acta Crystallogr D Biol Crystallogr. 2003 Dec;59(Pt 12):2356-8. Epub 2003 Nov 27. Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. Holyoak T1, Fenn TD, Wilson MA, Moulin AG, Ringe D, Petsko GA. Best, Wally -- Walter R.P. Novak Associate Professor and Chair of Chemistry Wabash College 301 W. Wabash Ave. Crawfordsville, IN 47933 Phone: 765-361-6407 Fax: 765-361-6149 Date:Tue, 23 Oct 2018 13:29:10 -0700 From:Kevin Jin Subject: Re: suggestions for cryoprotectant Dear Firdous, Probably other people already suggested above, and I don't know which kind of protein in your case. Here is my suggestion, you may try sugar solution or polysaccharide solution as cryoprotectant. The whole idea is to provide alternative H-bond network to support protein freezing. Indeed, some startups are using similar ideas to develop cryoprotectants for medical applications. Best, Kevin On Fri, Oct 19, 2018 at 2:58 PM Firdous Tarique wrote: > Dear members > > I have got beautiful crystal hits in SaltRx screens which are not > diffracting to a good resoultion. All of them are salt based condition and > I am not able to formulate a good cryoprotectant for these crystals. I also > think that in my case the poor resolution is due to a poor cryoprotectant > selection. > > The conditions are as follows: > > 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0 > 2>0.5M KCN 100mM Tris pH8.5 > 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0 > 4>4M Sodium Nitrate 100mM Tris pH8.5 > 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6 > > There are few more conditions but so far not able to see good diffraction > with using lower peg and glycerol based cryoprotectants. > > Can anybody suggest me good cryos conditions for salt based > crystallization conditions or anything good for SaltRx crystallization hits. > > Thanks > > Firdous > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > -- Kevin Jin Sharing knowledge each other is always very joyful.. Website: http://www.jinkai.org/ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
[ccp4bb] RapiData 2019
Dear all, Applications are now open for the 2019 edition of RapiData at SSRL. RapiData is a practical course in macromolecular X-ray diffraction data collection, data processing and structure solution. The aim of the RapiData course is to educate and train young scientists in data collection and processing methods at synchrotron beamlines, using state-of-the-art software and instrumentation. The course will be held at the Stanford Synchrotron Radiation Lightsource (SSRL) located on the SLAC National Accelerator Laboratory campus in Menlo Park, CA, from May 5-10 2019. Course organizers for 2019 are Silvia Russi, Clyde Smith and Jeney Wierman. The course will comprise hands-on experiments at the SSRL beamlines, software tutorials, and lectures on the following topics: Specimen preparation, Data collection strategies, X-ray light sources, X-ray detectors, Data reduction, Structure solution by MAD, SAD and Molecular Replacement, Complementary methods (spectroscopy and small angle scattering) Please visit http://smb.slac.stanford.edu/news/rapidata/rapidata-2019/ and use the links on the "registration" page to apply for the course. The application deadline is December 31 2018. Successful applicants will be notified early in January 2019 and invited to register and book accommodation at the SLAC Guesthouse at that time. A limited amount of travel support funding may be available. Please direct questions to Silvia Russi (sru...@slac.stanford.edu), Jeney Wierman (jwier...@slac.stanford.edu) or Clyde Smith (csm...@slac.stanford.edu). To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
[ccp4bb] Refmac jelly body parameters
Dear Refmac developers, I am trying to reproduce some successful refmac runs but cannot get the previous results despite playing around with the parameters. I did those runs in Feb 2014 (Refmac 5.8.0049) but unfortunately have overwritten the log files in a backup. I still have the pdbs, mtz and my notes. I was using jelly body and Prosmart at a resolution of 3.1 A, which worked superb and I could track some unexpected conformational changes. In several successive runs each with 100 cycles Rwork and Rfree were decreasing slowly and continuously and finally showed two rigid body movements. When I now repeat those runs (same pdb, same mtz input files and using the prosmart restraint file from the former runs) I get very similar Rwork/Refree but not the rigid body movements of these domains again in the resulting pdb. If I loosen the restraints in jelly body Rwork decreases but Rfree stays, indicating some overfitting. And also in the resulting pdbs the previously observbed conformational change is not observed. I tried also to increase the number of cycles to some insane numbers (>600), but this did not improve the situation. Any ideas what might go wrong? I am really fond of jelly body and would like now to check a series of new data sets at low resolution for the extent of the observed (and verified) conformational change. Best regards, Guenter To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
[ccp4bb] Post-Doc Opportunity
Hello All, I have an Post-Doc opening in my lab for an X-ray crystallographer to support our drug development efforts. Please see the ad (https://jobs.sciencecareers.org/job/485983/post-doctoral-research-associate-in-x-ray-crystallography/) for more information or contact me at dfla...@purdue.edu if you have any questions. Cheers, Dan To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
[ccp4bb] Postdoctoral position in artificial intelligence
I am forwarding this for my brother: https://job-search.astrazeneca.com/job/gothenburg/postdoc -fellow-in-artificial-intelligence/7684/9616027 *** Markus Seeliger Associate Professor Department of Pharmacological Sciences Stony Brook University Medical School, BST 7-170 Stony Brook, NY 11794-8651 office: (631) 444-3558 lab: (631) 638-1299 fax: (631) 444-9749 https://www.pharm.stonybrook.edu/markus-seeliger-lab-welcome markus.seeli...@stonybrook.edu *** To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
Re: [ccp4bb] Should I optimize these crystals?
Hi Marta, 1-- There is always a possibility of forming detergent/lipid (cholesterol in your case) -containing crystals combined with other small molecule(s) (organic or not). Although you did not specify the width of the oscillation used to collect the frame displayed in your mail (was it 45 degrees or 0.5 degrees?) I see signs of a lattice at least on the vertical direction (3 rows can be distinguished it seems), can you derive a "cell-dimension" along that direction? 2- If you have access to a fluorescence-coupled light microscope (like a a PRS-1000 Korima instrument) maybe you could see if your crystals glow (provided your proteins contain tryptophans). Crystals from drop 1 seem better candidates (they look 'thicker') for a fluorescence scan, the ones from drop 2 are really thin and curvy, so it might be difficult to see in that case. This could help you characterizing and ruling things out more easily. 3- You mentioned that you analyzed the content of washed/dissolved crystals by silver-stained SDS PAGE. Forgive me for asking if you also analyzed the last wash step on this sivler-stained SDS PAGE gel to rule out that the protein signal you observed was not cross-contamination by a carry-over effect. Good luck. Good luck On Wed, Oct 24, 2018 at 1:31 PM marta borowska wrote: > Dear all, > > I have grown crystals of a membrane protein complex, that I initially > verified on SDS-PAGE. These crystals grew only if membrane protein > component is there. The condition is polypropylene glycol 400, > cryoprotected with ethylene glycol, sample buffer has 150mM NaCl on top of > detergent with cholesterol. These long thin needles are quite sturdy and > either didn't diffract or diffracted like small molecule (I cannot exclude > the possibility of contamination stuck around crystal). After the > synchrotron trip harvested crystals were washed (most did not dissolve in > Urea or NaOH!), dissolved in 10% mild detergent and still showed protein > from 4-6 crystals when analyzed on Silver Stain SDS-PAGE. > > I would appreciate your input on whether some of you encountered similar > patterns and if you think I should proceed with more optimization on these > crystals. > > Thank you, > Marta > > > -- > > Marta T. Borowska > > Graduate Student > > Adams Lab > > Department of Biochemistry and Molecular Biology > > The University of Chicago > > 929 E. 57th Street > > Chicago, IL 60637 > > Lab: GCIS W229 > > Lab phone: 773-834-0660 > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pegea at mednet.ucla.edu To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
[ccp4bb] chapronin request
Hi, Sorry for unrelated email. We are working on a protein however much of it is unfolded when expressed in *E coli*. I was wondering if some one can help us by providing chapronin constructs (DNA J and K, GroEL, GroES or any other). Many thanks! Regards Dr Muhammad Imran Assistant Professor Biological Sciences Department Forman Christian College A Chartered University Lahore. Office: Armacost Science Building Room S-337, Ext: 552 Mobile: 0092-333-9990735 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
