Dear Deepak, As some will have already said a good first check is to X-ray the crystals at the temperature they were grown at. This is not too hard and there are numerous approaches available. Some synchrotron sources, e.g. SSRL, have even enabled this as a remote capability. This can tell you if the initial crystal quality is poor or if your cryoprotectant conditions are damaging the initial crystal. If they are, there are various approaches to introduce cryoprotectants slowly or even increase the cryoprotectant conditions in the initial screen slightly. If you plan to use glycerol I would use a small % in the initial solution. You do seem to have crystalline ice from your diffraction images.
A very successful and easily implemented approach is seeding. There is quite a bit of literature out there and commercial low-cost kits like the seed bead at Hampton-Research that can help this. Our Structural Biology Resources page at https://hwi.buffalo.edu/structural-biology-resources/ has links to papers in this area under the Crystallization Resources section. There may be other advice there also. In our Crystallization Center (http://getacrystal.org) we would typically optimize several chemically distinct conditions if multiple initial crystal hits were seen. It’s always better to do that rather than focus on a single condition. As others have said, a microfocus source and rastering over the needle-like crystals you have may produce some results. More complex, but also seen to be successful in cases, is altering the humidity of the crystals. There are commercial devices to do this and beamlines equipped with humidity control. This is a more technically challenging approach. Good luck, Eddie Edward Snell Ph.D. President and CEO | Hauptman-Woodward Medical Research Institute Director | NSF BioXFEL Science and Technology Center Professor, Materials Design and Innovation | University at Buffalo, SUNY p: +1 716 898 8631 | f: +1 716 898 8660 e: [email protected]<mailto:[email protected]> skype: eddie.snell Hauptman-Woodward Medical Research Institute 700 Ellicott Street | Buffalo, NY 14203-1102 hwi.buffalo.edu<https://hwi.buffalo.edu/> [hwi-logo-primary-horizontal] From: CCP4 bulletin board <[email protected]> On Behalf Of Deepak Deepak Sent: Tuesday, May 18, 2021 6:08 AM To: [email protected] Subject: [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&nbs Caution! This message was sent from outside your organization. sophospsmartbannerend 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 ######################################################################## 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/
