Hi, three things come to mind besides what has been mentioned already
- many proteins that have an affinity for nucleic acids actually love sulfates or phosphates… And yes, phosphates may lead to salt crystals at higher pH values but this should not stop you a priori from screening with the same (conditions under-represented in most screens…) - what is the pI of your protein? Is it very basic? If so, apply lessons learned from the Hofmeister series on ions and their effect on protein stability and solubility. Solubility of basic proteins (pI > 9) can be improved by adding some ammonium sulfate (100-200 mM in the protein buffers) and pay special attention to crystal screening solutions/conditions with thiocyanate, nitrate and chloride as these are good ions that stabilise basic proteins (and thereby promote crystal formation) - what does a sequence comparison shown with homologues that are in the database? Does the protein by chance have long(er) N and/or C- termini or a large loop? Limited proteolytic digestion of the protein followed by SDS-PAGE analysis will be helpful in identifying disruptive factors. Good luck, Jeroen __ Dr. math. et dis. nat. Jeroen R. Mesters Biological Safety Officer (BBS) Deputy, Lecturer, Program Coordinator Infection Biology Visiting Professorship in Biophysics - University South Bohemia University of Lübeck Center for Structural and Cell Biology in Medicine Institute of Biochemistry Ratzeburger Allee 160 23562 Lübeck Tel +49 451 3101 3105 https://orcid.org/0000-0001-8532-6699 Am 10.01.2025 um 02:00 schrieb Marco Bravo <0000d0eb7bee83ae-dmarc-requ...@jiscmail.ac.uk>: Hello all, I am seeking some advice or ideas to crystalize a nuclease protein. It exists within a heterodimer, homodimer, and maybe monomer as well depending on the species from which the protein is in. The protein has been shown to have nuclease activity on its own depending on the species. I am screening the protein from three different species and have setup so many crystal high-throughput crystal screens with and without DNA. I never get protein crystals for these proteins, I have done thermal shift assays to identify a buffer that stabilizes the protein better than the storage buffer and found some conditions that increased the melting temp by 4-5 degrees. I screened the protein again in these new stabilizing buffers but still have not gotten any hits. There are protein structures for the protein in the pdb with its partner protein in the heterodimer formation. I have experience screening other proteins and getting several hits per kit so I know what to expect for a successful screen. Any advice would be helpful, I want to retain the full-length protein for structural studies. I would appreciate any ideas or advice for moving forward and trying to obtain crystals. Thanks! Marco ######################################################################## 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/ ######################################################################## 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/
