> > is it logical to say that if the protein is highly charged (either > negative or positive), it is likely to be more soluble and resist > crystallization due to electrostatic repulsion?
Hi Kavyashreem The project that I mentioned, where we looked at the crystallization conditions reported in the PDB, also looked at the areas of amino acids (and atoms) on the surfaces of proteins and tried to correlate the various groups with crystallization conditions. Positive, negative, charged, polar or hydrophobic groups had very little effect on the concentration of precipitant (we looked at PEG and ammonium sulfate) and seemed not to affect the choice of precipitant (looking at all precipitants). The only thing you could say was that the area of all charged and polar groups as a proportion of the total surface area was roughly constant. If the charged/polar area was high, the protein was crystallized at slightly higher protein concentrations, and slightly higher concentrations of precipitant were used on average. But it should be possible to make predictions. DeepMind is probably working on this right now! Best wishes, Patrick On Mon, Feb 5, 2024 at 3:06 PM kavyashreem <kavyashr...@instem.res.in> wrote: > Dear all, > > Thank you all for your valuable experiences, inputs and references!! I > shall try them and hope for some good news! Its good to know there are so > many examples of crystallization at such high concentrations. > > A curious question - is it logical to say that if the protein is highly > charged (either negative or positive), it is likely to be more soluble and > resist crystallization due to electrostatic repulsion? Our protein has > highly positively charged surface, although with some small negative > patches. > > Following are some of the suggestions indicated: > > 1. Use water (will try) > > 2. Change buffers, pH temperature - (done) > > 3. Seeding (will try) > > 4. Methylating lysines (will try!!) > > 5. Surface entropy reduction (ongoing) > > 6. Optimize constructs (done) > > 7. Different ratios (done) > > 8. Keep concentrating (will try, the problem is yield is low!) > > 9. High salt concentrations (will try) > > 10. Organic solvents (though about it) > > 11. Mutations (ongoing) > > Thank you > > Regards > > Kavya > > On 2024-02-05 15:57, kavyashreem wrote: > > Dear All, > > Has anyone worked on a protein which is highly soluble even at 80mg/ml? > > We have one such candidate, which does not precipitate even at 80mg/ml > instead forms phase separated globules in crystallization plate, which > eventually hardens over a period of 1 to 1.5 months (which is florescent > under UV microscope.) > > We tried screening at different pH, but failed to get any hits. > > Since we got few conditions in which the phase separated globules > solidified, we focused on them and expanded with 120mg/ml protein, still > there were not visible precipitates except for the phase separation. This > has been a challenging target so far. We have tried with different > constructs, which unfortunately are not soluble! > > Does POMs help in such cases? Or do you have any other suggestion. > > Thank you > > Regards > > Kavya > > > > > *CONFIDENTIAL: This email and any files transmitted with it are > confidential and intended solely for the use of the individual or group to > whom they are addressed. If you have received this email in error, please > notify the sender immediately and delete this e-mail from your system. It > is NOT AUTHORISED to copy, forward, or in any way reveal the contents of > this message to anyone.* > > > > > *CONFIDENTIAL: This email and any files transmitted with it are > confidential and intended solely for the use of the individual or group to > whom they are addressed. If you have received this email in error, please > notify the sender immediately and delete this e-mail from your system. 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