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
>>>
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