Dear Guillaume, That is right, if not for cations it would not have been possible.
We will check with the surface entropy reduction. Since we are designing inhibitors for it, we cannot afford to do to much modifications. Thank you Regards Kavya On 2024-02-06 03:49, Guillaume Gaullier wrote: > Hello, > > Regarding this question: > >> 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. > > A counter example coming to mind is DNA: it is very strongly charged, yet has > been crystallized many times (you can find multiple examples of DNA-only > crystal structures in the PDB). Actually charges arranged so regularly along > the molecule actually help crystallization, when using an adequate > concentration of divalent cations. > Since fusion to a crystallizable scaffold protein has been suggested, I would > like to add that you could take inspiration from this paper from David > Baker's lab to design the scaffold protein (instead of using MBP or similar > ones): https://doi.org/10.1038/s41563-023-01683-1 > Pretty crazy things in there: spontaneous crystallization upon mixing E. coli > lysates, crystals that survive being autoclaved... If you know somebody who > knows how to use Rosetta for de novo computational design, this might be > worth a try. > An alternative would be to design de novo a high-affinity and crystallizable > binder for your protein. Similarly as the fusion protein approach, de novo > design may or may not be easier than alternatives, depending on protein > design expertise near you. > > I hope this helps, > > Guillaume > > On 5 Feb 2024, at 22:01, Tao-Hsin Chang <taohsin.ch...@gmail.com> wrote: > > Dear Kavya, > > I wanted to share with you that we have faced the same issue with a few > projects. In addition to the great suggestions given earlier, I recommend > trying out fusion proteins, such as the surface entropy reduction MBP mutant, > or using protein binding partners like antibodies or natural binders. These > strategies can help alter the protein properties and facilitate new crystal > contacts. Additionally, it may be worth experimenting with reduced salt > concentrations or using a salt-free buffer, akin to using water in place of a > buffer. > > PMID: 32541044 > PMID: 26850170 > > Wishing you the best of luck, > Tao-Hsin Chang > > On Feb 5, 2024, at 10:05 AM, 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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