After taking images at room temp and assuming good diffraction then, I would kick up the PEG400 concentration and as a first go on the PEG400 condition and try low molecular weight PEG's as a cryo protectant for the other two conditions.
Good Luck, been there and have exposed many crystals in the search of a good cryo. Len Leonard M. Thomas Ph.D. Macromolecular Crystallography Laboratory Department of Chemistry and Biochemistry University of Oklahoma 101 Stephenson Parkway Norman, OK 73019 405-325-1126 [email protected] http://www.ou.edu/structuralbiology/cobre-core-facilities/mcl ________________________________________ From: CCP4 bulletin board <[email protected]> on behalf of Saurabh Upadhyay <[email protected]> Sent: Sunday, March 21, 2021 1:19 PM To: [email protected] Subject: Re: [ccp4bb] Suggestions to improve resolution of protein crystals Dear Everton and Nukri, Thank you for your kind reply, we were also speculating that there could be some issues with cryoprotection. We have used two cryoprotectants naming; Parabar (10312) and Paraffin Oil from Hampton Research company. We have also used glycerol and respective mother liquors as well. However, there were no improvements in the diffraction pattern or resolution. Can you suggest some modifications regarding cryoprotectants or some ambient temperature for cryoprotection. Kusuma School of Biological Sciences, Indian Institute of Technology, Hauz Khas, New Delhi-110016, India On Sun, 21 Mar 2021, 23:42 Nukri Sanishvili, <[email protected]<mailto:[email protected]>> wrote: Dear Saurab, It's a shame these crystals do not diffract well - they look so good! I assume the diffraction you saw was recorded at cryo temperatures? If so, it is possible that you may be damaging the crystals during cryo-pretection and cryo-cooling. Before you go too deep into changing the crystallization conditions, I would suggest taking a shot or two at ambient temperatures and see if the diffraction is any better. If so, you may need to optimize the cryo protocol and not the crystallization conditions. Regards, Nukri On Sun, Mar 21, 2021 at 1:04 PM Saurabh Upadhyay <[email protected]<mailto:[email protected]>> wrote: Dear All, I am trying to crystalize a protein, which occurs in monomeric (60kDa) and tetrameric (240kDa) form. The protein during crystallization was in MES buffer pH-6 and the method of crystallization was "Sitting drop" in "Proplex" condition. Some of the conditions in which crystals were obtained are: 1) 0.1M HEPES pH-7; 10% (w/v) PEG 4000 2) 0.1M Tris, pH-8; 8% (w/v) PEG 8000 3) 0.1M Tris, pH-8; 25% (v/v) PEG 400 For reference, I have attached the images of the crystals observed below. However, the diffraction pattern obtained and the resolution of crystals were very poor. Even some mosaicity has been observed. Kindly suggest some methods or modifications, to improve the resolution and diffraction pattern of the crystals obtained. Thanking You, Sincerely, Saurabh Upadhyay, Ph.D. Scholar c/o Dr. Ashok kumar Patel, Kusuma School of Biological Sciences, Indian Institute of Technology, Hauz Khas, New Delhi-110016, India ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_WA-2DJISC.exe-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=qKdtBuuu6dQK9MsRUVJ2DPXW6oayO8fu4TfEHS8sGNk&r=LYFS_Pt-UDexWUsCj6bDwQ&m=YP2Y18IUvj2kCwWWvqngR3x8j5FgTPUin1rQ-a4pCa0&s=Dole2AfBZBVEMPWMCsSBlRJ2VLdr9Jb_jdSM8YCc3tU&e=> ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1<https://urldefense.proofpoint.com/v2/url?u=https-3A__www.jiscmail.ac.uk_cgi-2Dbin_WA-2DJISC.exe-3FSUBED1-3DCCP4BB-26A-3D1&d=DwMFaQ&c=qKdtBuuu6dQK9MsRUVJ2DPXW6oayO8fu4TfEHS8sGNk&r=LYFS_Pt-UDexWUsCj6bDwQ&m=YP2Y18IUvj2kCwWWvqngR3x8j5FgTPUin1rQ-a4pCa0&s=Dole2AfBZBVEMPWMCsSBlRJ2VLdr9Jb_jdSM8YCc3tU&e=> ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
