[ccp4bb] Workshop: Directed Evolution Approaches in Structural Biology
Directed Evolution Approaches in Structural Biology Wed 30th Thu 31st January 2008 at EMBL Grenoble, France. Funded by: SPINE2COMPLEXES TEACH-SG (EU FP6) Info Register: www-db.embl.de/jss/EmblGroupsOrg/conf_91 See also: www.spine2.eu/SPINE2/meetings/index.jsp?m=28 A number of laboratories are now developing or using library-based strategies in combination with structural biology. This may be to overcome problems in the structure solution process (expression, crystallisation etc.) or to study/engineer proteins for structural and functional studies. We aim to strengthen links between these labs and provide training for scientists interested in this field. Topics 1. Library strategies for “difficult-to-express” proteins 2. Scaffold proteins and binders as cocrystallisation chaperones 3. Combinatorial approaches to protein complex definition and expression 4. Structural studies of in vitro evolved proteins ·Colony filtration (CoFi). ·Combinatorial Domain Hunting (CDH). ·Efficient fragmentation, truncation mutation approaches ·Robotics truncation libraries. ·GFP solubility screening. ·Screening by FACS and fluorescent colony picking. ·DARPins ribosome display. ·DNA shuffling/Oil-in-water emulsions. ·Use of display technologies Speakers Stephanie Cabantous Waldo Lab, Los Alamos, USA Thomas HuberPlückthun Lab, University of Zurich Speaker Nordlund Lab, Karolinska Institute, Sweden Renos Savva Domainex Ltd, UK Darren Hart EMBL Grenoble, France Martin WalshMRC France, Grenoble Amir AharoniWeizmann Institute, Israel Sabine Mazaleyrat AstraZeneca (ex), UK Chris Meier UCB-Celltech, UK -- ** Dr. Darren Hart, Team Leader High Throughput Group Grenoble Outstation European Molecular Biology Laboratory (EMBL) ** http://www.embl.fr/groups/htt/expression.html Email: [EMAIL PROTECTED] Tel: (33) 4 76 20 77 68 Fax: (33) 4 76 20 71 99 Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex 9, France **
Re: [ccp4bb] insoluble ligand
Dear Simon, DMSO concentrations lower than 5% usually do not alter crystallizability of a protein. In case you want to avoid this solvent may I suggest you trying out two methods that worked for me. 1. If you grow crystals in PEGs or similar molecules you might try to solubilize the compound in a small amount of precipitant solution. This is helpful if you want just a 1:4 protein:ligand ratio. 2. Sometimes solubility is low even in 5% DMSO (or diluted solutions of glycerol, alcohols and similar molecules). In these cases setting up drops in the presence a saturated solution and some precipitate of the compound may also lead to good co-crystals. As one molecule passes from the saturated solution to the bound state, a new molecule is solubilized from the precipitate, which gradually dissolves and passes from the solution to its binding site in the protein. Indeed, this sounds like a soaking experiment and it works well if the compound is coloured, so that you can see if the crystals actually become of the same colour. Just remember to wash them thoroughly before measurements, in order to remove traces of the ligand precipitate that would result in bad diffraction pattern. Hope this will help you. Marco
Re: [ccp4bb] insoluble ligand
Simon, We routinely obtain structures from protein solutions with a big pellet of ligand in the bottom of the tube. For co-crystallizations we add 1mM compound to a 0.3mM solution of the protein and incubate overnight. Many of the compounds are only soluble to 50micromolar, so we get a lot of precipitate. The next day, we spin the tube at high speed, and use the supernatant for crystallization trials. We have started from 100mM stocks in 100% DMSO or ethanol. This has worked for compounds ranging for picomolar to micromolar affinity, which surprised us, but it worked. Regards, Kendall On 12/11/07 11:55 AM, Yue Li [EMAIL PROTECTED] wrote: Hi all, I have one ligand which is insoluble in water, and I would like to co-crystallize it with my protein. Is there any other method except for dissolving it in DMSO ? Thanks Simon
[ccp4bb] protein precipitated when they formed a complex
Dear All, Recently I posted a question about protein induced protein precipitation. Firstly I'd like to thank many folks for their good ideas. Later on I did a titration experiment with one protein concentration fixed at 0.4mg/ml(about 10uM). Now it is clear that these two proteins stoichiometrically precipitated when they formed a 1:1 complex. The excess of individual proteins was just soluble in the buffer. How come these two proteins co-precipitated when they formed a complex? Does anyone know some methods to keep the complex soluble enough for crystallization? By the way, there is some additional information about the individual components. One has a pI of 6.5, and the other has a pI of 10. Any suggestions will be highly appreciated. Jerry McCully _ Get the power of Windows + Web with the new Windows Live. http://www.windowslive.com?ocid=TXT_TAGHM_Wave2_powerofwindows_122007
Re: [ccp4bb] upgrading ccp4 broke my Coot stereo
What is the operating system? These things are system-dependent... On Wed, 12 Dec 2007 15:42:39 -0500 Robert Grant [EMAIL PROTECTED] wrote: It would appear that an environmental variable involving a library path is missing or wrong but I have not been able to figure out what it is. Any ideas?
[ccp4bb] ccp4bb: ccp4i and ligand library
Hi, I've recently made a ligand using the Sketcher module in CCP4i and made the cif dictionary. I can run Refmac5 in review restraints mode with the dictionary in my working directory and it seems to work fine. However, now I am trying to refine my ligand in the protein structure and I get the error message New ligand has been encountered. Stopping now, even though I've already made the dictionary and have it selected in the Library blank. In the past I added the keyword Make Check None to my script file to get things working. I've become found of the GUI-- is there a special trick for getting the GUI to use my dictionary? Thanks for your help! Catherine Regni, Ph.D. - Never miss a thing. Make Yahoo your homepage.
Re: [ccp4bb] protein precipitated when they formed a complex
Dear Jerry, One way I can think of would be to try the magical polymer NV-10 sold by Novexin (UK). It does actually work very well for proteins with low solubility. You'd add a few mg/ml of polymer to each protein solution, mix the two and see what happens. This polymer has already saved several of my poorly soluble proteins and I hear from others that it worked well for them, too. If you're desperate - I would recommend rational surface mutagenesis. We, and others, have designed surface mutants with greatly improved solubility :-) Or fuse with something nice and soluble. Artem _ From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Jerry McCully Sent: Wednesday, December 12, 2007 3:31 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] protein precipitated when they formed a complex Dear All, Recently I posted a question about protein induced protein precipitation. Firstly I'd like to thank many folks for their good ideas. Later on I did a titration experiment with one protein concentration fixed at 0.4mg/ml(about 10uM). Now it is clear that these two proteins stoichiometrically precipitated when they formed a 1:1 complex. The excess of individual proteins was just soluble in the buffer. How come these two proteins co-precipitated when they formed a complex? Does anyone know some methods to keep the complex soluble enough for crystallization? By the way, there is some additional information about the individual components. One has a pI of 6.5, and the other has a pI of 10. Any suggestions will be highly appreciated. Jerry McCully _ Get the power of Windows + Web with the new Windows Live. Get it now! http://www.windowslive.com?ocid=TXT_TAGHM_Wave2_powerofwindows_122007
[ccp4bb] upgrading ccp4 broke my Coot stereo
Forgot to include that it is a Red Hat EL4 OS. -- Forwarded message -- From: Robert Grant [EMAIL PROTECTED] Date: Dec 12, 2007 3:42 PM Subject: upgrading ccp4 broke my Coot stereo To: ccp4bb@jiscmail.ac.uk I recently upgraded from ccp4 version 5.99.5 to 6.02. In doing so I have lost the ability to run Coot with hardware stereo. When I fire up Coot it reports: Gtk-WARNING **: Unable to locate loadable module in module_path: libbluecurve.s o When I try to switch to hardware stereo it says: CATASTROPHIC ERROR:: in gl_extras no GtkGL widget! WARNING:: switch to hardware_stereo_mode failed The new version of ccp4 was downloaded along with the latest Coot, Phaser, TclTk, and Chooch. Nothing has changed in my /etc/X11/xorg.conf file, which I had to modify to get the stereo to work in the 1st place. It would appear that an environmental variable involving a library path is missing or wrong but I have not been able to figure out what it is. Any ideas?
Re: [ccp4bb] upgrading ccp4 broke my Coot stereo
I just noticed there is a space between s and o in .so: Robert Grant wrote: Gtk-WARNING **: Unable to locate loadable module in module_path: libbluecurve.s o Probably a mistake in pasting the error message, but if not it could be significant. Ed
