Hello Marco This is a classic case of protein that just won't behave :)
Nil desperandum! Without rewriting several large ish chapters on protein crystallization, let's review your options from a bird's eye view: 0. Modify crystallization setup This is something that you already have explored cocktail consitions quite a bit, but there are still many possible solutions left - such as temperature, horse hair, pickle juice, magnetism, vibration, laser light, dust from the shelf, change in crystallization method (dialysis versus sitting versus hanging drops, etc.), homo and heterologous seeding - all the above have been used successfully to crystallize proteins, you can find references to all of this and much more... 1. Modify existing sample: chemical modification (methylation, acetylation, heavy atom treatment), physical modification (gently cooking the sample at sub denaturing temperatures to denatured all partially broken molecules), or enzymatic modification (proteolysis or ligation, etc. I guess this technically falls under chemical modification but hey this is just an email). You have already tried changing buffer, but you also should consider concentration effects and stabilizing/destabilizing additives. If you are convinced that DNA is involved, consider crosslinking the DNA to the protein. 2. Modify construct and re-express. This is a broad swath of options that include chunking, termini or loop truncation, or crystallogenic mutations, as well as fusion of crystal friendly guest proteins like mbp, t4l, try, etc). You mentioned the desire to keep FL protein intact, but can you at least remove some dangling bits, for starters? 3. Prayer to the higher authority (I recommend praying to Cosmic Megatron, the Party God from adventure time, Reviewer #3, or perhaps the flying spaghetti monster; all these options should work equally well. There are dozens of possible options and it is hard to say what might work for you specifically without more details. Artem On Thu, Jan 9, 2025, 20:01 Marco Bravo < 0000d0eb7bee83ae-dmarc-requ...@jiscmail.ac.uk> wrote: > 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/
