Hello and you are welcome. - You can add things after you purify, or buffer exchange on a concentrator
- sometimes one residue change makes a difference, removing 20 is almost certainly going to change things, maybe for the better - fusion of guest domains was mentioned with the intent of crystallizing the fusion-but you should pay attention to linker design to avoid too much flexibility - Protein crosslinking to DNA is not easy, I can recommend making special DNA with suitable covalent handles (SH, NH2, click, iodoacetic, etc.) and possibly introducing paired opposite handle on the protein. https://www.nature.com/articles/ncomms6849#ref-CR19 https://academic.oup.com/nar/article/28/2/e4/1039690?login=false https://www.ncbi.nlm.nih.gov/books/NBK7107/ https://home.ccr.cancer.gov/csb/nihxray/Tips-and-Tricks_Crystallization.html Hard to pack 20+ years of crystallizing things into a few emails :) Artem On Thu, Jan 9, 2025, 20:50 Marco Bravo <marco.br...@email.ucr.edu> wrote: > Hi Artem! > Thank you for the suggestions, do you have recommendation of common > protein-DNA crosslinking methods for crystalization? I suppose I also > Identified de-stablizing buffers with my thermal shift assays but these > have been hard to work with as they do indeed destablizie the protein > leading to issues of zero elution from the gel filtration column. I have > been thinking of truncating just the first 20amino acid residues from the > N-terminus but am unsure if this will make a difference in > crystallization. I suppose a fusion tag like MBP is worth a shot but > should I keep the tag on during crystalization screening? I know I asked a > lot of questions, this also helps me think of how to move forward ! Thank > you! I will explore some of these ideas! > > Marco > > On Thu, Jan 9, 2025 at 5:39 PM Artem Evdokimov <artem.evdoki...@gmail.com> > wrote: > >> 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/
