Herman's suggestion is very good. But I'd go one step further and try to grow crystals in 30-40% PEG400. Then you definitely do not need any additional cryoprotectant. There is a chance that your crystals are of poor quality to start with. In that case no cryoprotection will help. You can test that by collecting few diffraction images at ambient temps. If that will prove true that your crystals are not good enough then either that particular crystallization condition is not suitable for your complex or, most likely, more attention should be paid on getting more homogeneous protein solution. For more project oriented advice you may contact off list.
Vaheh Oganesyan, Ph.D. [cid:[email protected]] R&D | Antibody Discovery and Protein Engineering One Medimmune Way, Gaithersburg, MD 20878 T: 301-398-5851 [email protected]<mailto:[email protected]> From: CCP4 bulletin board <[email protected]> On Behalf Of Schreuder, Herman /DE Sent: Tuesday, May 18, 2021 8:33 AM To: [email protected] Subject: [ccp4bb] AW: [ccp4bb] sugestions on weak diffracting protein crystals Dear Deepak, the cryoprotection may destroy the diffraction. I would also try diffraction at room temperature, using special sleeves to prevent dehydration. With 20% PEG400, you could also try to freeze the crystals without using additional cryoprotectant. If you are lucky, it may work. Best, Herman Von: CCP4 bulletin board <[email protected]<mailto:[email protected]>> Im Auftrag von Deepak Deepak Gesendet: Dienstag, 18. Mai 2021 12:08 An: [email protected]<mailto:[email protected]> Betreff: [ccp4bb] sugestions on weak diffracting protein crystals Dear all, I have got multiple crystals (see picture 1) of a protein (8kDa) with a helical aromatic oligoamide foldamer (5kDa) but these crystals diffract very poorly (see the diffraction pattern in picture 2). I prepare a 1.3mM:1.3mM complex of protein: foldamer in 20mM Tris, pH 7.5 buffer. Crystals grew in 3-5 days in sitting and hanging drop at 20 Deg C and 25 Deg C in the following conditions: - 20% PEG 400, 0.1M MES pH 6.0 -20% PEG 400, 0.1M Sodium Cacodylate pH 6.0 Multiple cryo used were: -25%Glycerol in mother solution -30% glycerol in water -30%PEG 400, -35% PEG 400 -20% PEG 8000 + 40% PEG 400 mix Kindly suggest some methods/modifications on how can I improve the resolution and get better-diffracting crystals. Please let me know if you need more information. Kind regards, Deepak Ph.D. Student PS: The protein is a DNA binding protein and I have crystallized and solved the structure of this protein with its DNA partner and now I crystallized it with our foldamers but diffraction is not good. There are multiple structures of the Protein+DNA complex in literature but no apo-protein structure as the protein needs a binding partner to crystallize. We already have solution studies showing a good binding. ________________________________ 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://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1&data=04%7C01%7CHerman.Schreuder%40SANOFI.COM%7C49a9b7a09d664c4f081108d919e66f22%7Caca3c8d6aa714e1aa10e03572fc58c0b%7C0%7C0%7C637569299785900813%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000&sdata=ELgp9Msphh8z2i4NhA7D4NZKIZiDRx%2F5pjYtITVFBI0%3D&reserved=0> ________________________________ 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://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> ######################################################################## 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/
