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Your protein might be not homogenious - i.e. not chemically (I assume it is of high purity) but structurally. Typical example is a very flexible albumin that crystallizes overnight, makes large and beautiful crystals looking perfect, but...diffracting barely to 5A the most and on a good day. Another reasons could be the structural variations due to enzymatic digestion (some molecules are intake some are cleaved, human thrombin was famous for that), partial deamination, partial oxidation, etc. Analyze your conditions starting from expression and purification and see what can be the possible culprit - try to eliminate it. If you work with active enzyme and try to make a complex with inhibitor to stabilize it - add inhibitor to the FROZEN sample and than thaw it on ice. Reduce the number of steps in handling this protein. Good luck - :) Ewa Dr. Ewa Skrzypczak-Jankun Associate Prof. Urology Research Center Medical University of Ohio 3065 Arlington Ave. Toledo OH 43614 phone 419-383-5414 fax 419-383-3785 e-mail: [EMAIL PROTECTED] >>> "Christensen, Jeff" <[EMAIL PROTECTED]> 11/28/2005 2:23 PM >>> Hi, Orly, If you're getting big crystals overnight that don't diffract well, my first thought is to try and slow down the growth of the crystal. If the faces of the crystal grow at different rates, rapid growth can induce stresses in the crystal that result in poor diffraction. Try diluting the protein sample 1:1 w/ nanoH20 and then set up vapor diffusion drops with protein:crystallant ratios of 1.5:1, 2.0:1, 2.5:1, etc. The larger volume of dilute protein will end up at the same final concentration as the drops your setting up now, but through slower protein concentration "in dropo" as the drop moves toward equilibrium. Also, have you tried mounting the crystals in a capillary rather than freezing, and then checking the resolution? This will tell you whether it's a problem inherent in the crystal or if your cryo-protectant is creating the problem. Good luck! Jeff Jeff Christensen Senior Research Associate deCODE biostructures 7869 NE Day Rd. W. Bainbridge Island, WA 98110 phone (206) 780-8933 fax (206) 780-8547 [EMAIL PROTECTED] ________________________________ From: [EMAIL PROTECTED] [mailto:[EMAIL PROTECTED] On Behalf Of Orly Dym Sent: Sunday, November 27, 2005 12:31 AM To: [email protected] Subject: [ccp4bb]: I have beautiful reasonably big crystals from a protein which diffract to 6A at the most. I have tried adding additives, crystallizing in different methods (hanging, drops, sitting drops and microbatch under oil). The crystals grow over night and contain high percent of water (they are very fragile). I also tried different methods of dehydration (using different PEG's, MPD, glycerol and so on). Currently I am trying to grow them at 4C. Any other ideas as to what I should try in order to improve the resolution? Thanks Orly Dr. Orly Dym Israel Structural Proteomics Center Department of Structural Biology Weizmann Institute of Science http://www.weizmann.ac.il/ISPC 972-8-934-3759 - Tel 972-8-934-4159 - Fax [EMAIL PROTECTED] On June 8, 2005, the Medical College of Ohio's name changed to the Medical University of Ohio. Please note that any contacts stored in your personal address book with the extension of mco.edu should now be changed to meduohio.edu to avoid any interruptions in email delivery. CONFIDENTIALITY NOTICE: This e-mail message, including any attachments, is for the sole use of the intended recipient(s) and may contain confidential and privileged information. Any unauthorized review, use, disclosure or distribution is prohibited. If you are not the intended recipient, please contact the sender by reply e-mail and destroy all copies of the original message. <<<<meduohio.edu>>>>
