[ccp4bb] need suggestions
Dear All, I have encountered one problem to optimization the crystallization condition manually. Actually i got the good shape and even size crystal in a screening plate. The condition are : 20% PEG 6000, 0.1M MES pH 6.0 whereas protein in 10mM Na-acetate pH 5.7 contain 50mM NaCl, 1mM EDTA &5mM BME. When I tried to optimize this on Linbro the drops are become heavily precipitate even a mints. I tried so many things to avoid this ppt such as start PEG conc from 7%, dilute protein at half conc., add more salt in a reservoir solution but no succeeds. The protein prep is same which was using to get the Hits. I would be very thankful if you people give me nice suggestions. Thanks Best Regards AFSHAN
Re: [ccp4bb] need suggestions
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Afshan, - - Can you reproduce the crystals in the Linbro plates with the identical solution used for the screening plate? - - Did you compare the composition of the Linbro plate vs. the screening plate? - - Did you check e.g. the pH of the original solution? - - Did you refresh the beta-ME / try a new protein prep? It goes off after some time, as far as I know. Regards, Tim On 04/16/2013 11:21 AM, Afshan Begum wrote: > > > Dear All, > > I have encountered one problem to optimization the crystallization > condition manually. Actually i got the good shape and even size > crystal in a screening plate. The condition are : > > 20% PEG 6000, 0.1M MES pH 6.0 whereas protein in 10mM Na-acetate pH > 5.7 contain 50mM NaCl, 1mM EDTA &5mM BME. When I tried to optimize > this on Linbro the drops are become heavily precipitate even a > mints. I tried so many things to avoid this ppt such as start PEG > conc from 7%, dilute protein at half conc., add more salt in a > reservoir solution but no succeeds. The protein prep is same which > was using to get the Hits. > > > I would be very thankful if you people give me nice suggestions. > > Thanks > > > Best Regards > > AFSHAN - -- - -- 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 Mozilla - http://enigmail.mozdev.org/ iD8DBQFRbSmWUxlJ7aRr7hoRAstnAKDlOMp84i3k4TxbzyPiXrdrmQmCUgCfWrLs ySGYCLJmu6yEJ7i94AEjqKQ= =EJjS -END PGP SIGNATURE-
Re: [ccp4bb] need suggestions
Hi Afshan, Have look at http://www.douglas.co.uk/Scaling_Up.htm, Patrick has some general tips on scaling up. Elsewhere on his site you'll find tips for conversion to microbatch, you may want to try that out as well. I assume the screen crystals were not big enough to mount? Flip Op 4/16/2013 11:21, Afshan Begum schreef: Dear All, I have encountered one problem to optimization the crystallization condition manually. Actually i got the good shape and even size crystal in a screening plate. The condition are : 20% PEG 6000, 0.1M MES pH 6.0 whereas protein in 10mM Na-acetate pH 5.7 contain 50mM NaCl, 1mM EDTA &5mM BME. When I tried to optimize this on Linbro the drops are become heavily precipitate even a mints. I tried so many things to avoid this ppt such as start PEG conc from 7%, dilute protein at half conc., add more salt in a reservoir solution but no succeeds. The protein prep is same which was using to get the Hits. I would be very thankful if you people give me nice suggestions. Thanks Best Regards AFSHAN
Re: [ccp4bb] need suggestions
Hi Afshan, You mention: The protein prep is same which was using to get the Hits. So how old is your prep ? Was it stored at 4C or in LN2? Do you know if your protein is stable under the conditions you stored it ? How long after purification did it take you to get those crystals ? How old is your screen ? MES goes bad as well over time. Jürgen .. Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://lupo.jhsph.edu On Apr 16, 2013, at 5:21, "Afshan Begum" wrote: > The protein prep is same which was using to get the Hits.
[ccp4bb] Need suggestions for protein: ligand ratio for co-crystallization
Hello everyone first I want to thank you guys in advance. I am trying to co-crystallize a 12 KDa protein with a 2 KDa peptide. The biochemistry studies showed the binding should be 1:1 . but the binding is not very strong ( Kd is around 20-40 uM). I am wondering, is there any bad effect of excessive peptide in the solution? someone suggested I use lot of peptide , say 3 times of the peptide to protein. usually what is the ratio you will try when you deal with this not-so-small ligand ? any suggestion is appreciated. Lei Feng
Re: [ccp4bb] Need suggestions for protein: ligand ratio for co-crystallization
Lei, 1. Consider making the complex and then purifying the excess peptide away by dialysis (size exclusion may be tricky since complex may be diluted in the process). 2. Conventional wisdom would be to try to minimize the amount of excess peptide as it may "interfere with crystallization". But can I tell you that if you have 3:1 peptide to protein ratio, excess peptide will inhibit crystallization? Not really, and in fact quite the opposite result is possible (won't say likely either way since I don't pretend to know how to crystallize proteins) - e.g. in some conditions complex may be disrupted by high salt content and you need excess ligand to keep it intact. Nobody knows what will work in each particular case - we only know general trends. If you have access to a crystallization robot - just try different ratios. If you don't, consider setting multiple drops on the same coverslip (with 22mm size four different color thick sharpies taped together in a rectangular arrangement make a great labeling tool). Cheers, Ed. On Tue, 2010-10-12 at 16:09 -0400, lei feng wrote: > Hello everyone > > first I want to thank you guys in advance. I am trying to > co-crystallize a 12 KDa protein with a 2 KDa peptide. The biochemistry > studies showed the binding should be 1:1 . but the binding is not very > strong ( Kd is around 20-40 uM). I am wondering, is there any > bad effect of excessive peptide in the solution? someone suggested I > use lot of peptide , say 3 times of the peptide to protein. > > usually what is the ratio you will try when you deal with this > not-so-small ligand ? > > any suggestion is appreciated. > > Lei Feng > -- "I'd jump in myself, if I weren't so good at whistling." Julian, King of Lemurs
[ccp4bb] Need suggestions on Mac Pro and accessories for structural biology work
Dear CCP4 community, I am posting to get your suggestions on a Mac Pro system for structural biology and not entertainment. Any comments will be appreciated. Please feel free to send off line messages if you prefer. My target is a Mac Pro with 8-12 cores and Dual AMD FirePro D700 GPUs. I will run structural biology programs with light to intense computation load, and 3D display of protein structures on a 24-27 inch monitor is a necessity. This system will be remotely connected to a cluster for time consuming computation. Here come the questions. 1. What will be a good monitor (brand, model, etc) for my setup? What specs should I pay attention when shopping for the monitor so I won't face any compatibility or 3D display issue? 2. Suggestions of emitters and glasses paired with Mac Pro and monitor are highly welcome. Or could I add an emitter on Mac Pro like on a Linux box? 3. There are many adapters and cables for Mac, so recommendations on adapter selection to make a Mac Pro-3D monitor package will be greatly appreciated too. 4. Anything I need to notice to have a complete ready-to-go system but is missed in this email (excluding mouse, keyboard, and speakers). Thank you for reading the post, and wish you a great start of the week. Best, Yanfeng
