Dear fellow CCP4bb'ers,
Generally one to two rounds of streak seeding can change a plate morphology into a shoebox morphology, but I have found that sometimes the reason for the plates in the first place is non-homogenity of your sample. Have you checked the sample by SDS and Native PAGE, and IEF gel also? You may be co-purifying a protease in your sample that is slowly chewing up your protein before it crystallizes. Do you have enough plates (10 or so of decent size) to harvest? You can wash all free protein off the crystals and then run a MS analysis of what actually crystallized. This can illuminate potential problems in your sample. Alternately, you can keep a sample (~30uL @ 5-10mg/mL) in the incubator next to the plates for the amount of time to crystallize, then run MS on the sample and see if it has changed. Alternately, if you are confident in the quality and purity of your sample, Artem Evdokimov has a useful protocol for harvesting fragile crystals at http://www.xtals.org/pdfs/needles.pdf. At least he used to, my IT department is blocking my access right now. I wish you the best of luck in your attempt to improve your crystals. Kind regards, Bryan From: CCP4 bulletin board [mailto:[email protected]] On Behalf Of Raji Edayathumangalam Sent: Monday, April 01, 2013 3:57 PM To: [email protected] Subject: Re: [ccp4bb] plate crystal optimization I hate to take you back to the step of crystallization but have you tried seeding to improve the crystal morphology/volume? If tinkering around with cryoprotectants and mounting techniques does not help your case, seeding might be worth considering. In some of the cases I've seen, my labmate was able to "transform" plates into 3D crystals. Of course, if there's good reason one is getting plates in the first places, seeding may not help. Just to keep that in the back of your mind given the magical "transformations" I've witnessed. Good luck! Raji On Mon, Apr 1, 2013 at 11:19 AM, Phoebe A. Rice <[email protected]> wrote: Among many possible reasons, the streaking could be caused mechanical stress to the thin plates during mounting. Have you tried those loops that look like miniature tennis rackets? Phoebe ________________________________ From: CCP4 bulletin board [[email protected]] on behalf of Rakesh Chatterjee [[email protected]] Sent: Monday, April 01, 2013 10:01 AM To: [email protected] Subject: [ccp4bb] plate crystal optimization hello everyone, i have a protein which co-purifies with a ligand (small molecule ~ 810 Dalton). i didn't know what kind of ligand is it so i am planing to identify it. meanwhile my protein crystallized as plates and while diffracting i found that the crystal is showing a good diffraction pattern upto 2.6 Angstroms in home source Cu K-alpha. however when the crystal rotates between 90 degrees to 135 degrees, the spots become streaky. should i try different cryo-MPD/ PEG400 etc? to circuvent the problem i did some additive screen but was not of much help. any valuable suggestion willbe handfull. thanx in advance rakesh -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University -------------------------------------------------------------------------- Confidentiality Notice: This message is private and may contain confidential and proprietary 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 unauthorized use or disclosure of the contents of this message is not permitted and may be unlawful.
