Re: [ccp4bb] [EXT] Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

Dear Patrick, Thank you for the insight! Regards Kavya On 2024-02-06 00:29, Patrick Shaw Stewart wrote: >> 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

Re: [ccp4bb] [EXT] Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

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

Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

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

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Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

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

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Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

> > 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

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Re: [ccp4bb] [EXT] [ccp4bb] Crystallizing a tough target

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

Re: [ccp4bb] [External] Re: [ccp4bb] Crystallizing a tough target

While I understand that you want to have protein concentration at its solubility limit, i had several proteins which can go to 60-80 mg/ml, they seems to get crystallized at 10-15 mg/ml. All you want is saturation in crystallization conditions. Dhiraj srivastava

Re: [ccp4bb] Crystallizing a tough target

For what it is worth, human serum albumin has been crystallized initially from 250 mg/ml solution back in ‘90s. When I started working on ternary complex hSA-FcRn-Fc (PDB Id 4N0U) was afraid that such high concentration couldn’t be achieved with amounts of FcRn I can reasonably express.

Re: [ccp4bb] Crystallizing a tough target

Dear Kavya, I'm sure there are several similar stories to learn from. I've worked with a point-mutant of a "normal" protein which made it thermostable - as revealed by DSF. It also resulted in an increased solubility, and the thing could be concentrated to 100 mg/mL and left on the bench for

Re: [ccp4bb] Crystallizing a tough target

HOD crystallised at 150mg/ml https://www.pnas.org/doi/10.1073/pnas.0909033107 Once I set the wrong time on the centrifuge and it went up to >200 mg/ml. I am sure many others have similar stories. All the best Roberto Roberto A Steiner www.steinerlab.org

Re: [ccp4bb] Crystallizing a tough target

Dear Kavya, Concanavalin A with glucoside involved 120 mg/ml protein. Details of the crystallisation are here:- https://doi.org/10.1016/0022-2836(87)90198-7 PDB code 1GIC. Best wishes, John Emeritus Professor John R Helliwell DSc > On 5 Feb 2024, at 10:27, kavyashreem wrote: > >  > Dear

Re: [ccp4bb] Crystallizing a tough target

Limited proteolytic might help, too. https://www.nature.com/articles/nmeth1118 Best wishes, Jon Cooper. jon.b.coo...@protonmail.com Sent from Proton Mail mobile Original Message On 5 Feb 2024, 10:27, kavyashreem wrote: > Dear All, > > Has anyone worked on a protein which is

Re: [ccp4bb] Error in "Prepare and validate files for deposition" task"

Dear Maria, Apologies for late reply. I'm not sure which version of "adding_stats_to_mmcif" is in CCP4 at the moment, but if you're facing the issue again in the future please could you try using the latest version at https://github.com/wwPDB/py-adding_stats_to_mmcif ? If it doesn't work,

Re: [ccp4bb] Crystallizing a tough target

Hi Kavya, You`r likely working with a very low PI sample, right? . Here are a few suggestions Final purification step at a pH closer to the pI:  Adjusting the pH during purification to be closer to the pI can reduce the solubility and promote precipitation. Non-PEG precipitants based kits:

Re: [ccp4bb] Crystallizing a tough target

Hi Kavya, In addition to other excellent suggestions, I would recommend trying reductive lysine methylation, which can make the protein more hydrophobic. PMID 17098187. Best wishes, Tomas On Mon, Feb 5, 2024 at 10:27 AM kavyashreem wrote: > > Dear All, > > Has anyone worked on a protein which

Re: [ccp4bb] Crystallizing a tough target

Hi, I have a small protein that is hugely soluble too. We could crystallize it in 3.5M ammonium sulfate with the help of a ligand that apparently triggered the crystallization. Try changing the temperature too. Good luck, GIA Le 05/02/2024 à 11:27, kavyashreem a écrit : Dear All, Has

Re: [ccp4bb] Crystallizing a tough target

Don't forget to try pure water. On 05/02/2024 10:44, Savvas Savvides wrote: Dear Kavya, we encountered this issue for a protein complex at 95 mg/mL involving Human Serum Albumin (HSA) and a designed protein binder (Alphabody) as described in Pannecoucke et al. 2021 Sci Adv 7 (13),

[ccp4bb] Re [ccp4bb] Crystallizing a tough target

Hi Kavya A few things I would try: -set up your plates (and incubate) at 4 degrees - perhaps in a cold room -use Alphafold model to identify patches of charge that you can mutate -perhaps even introduce a few sticky motifs (e.g. herein and ref 26

Re: [ccp4bb] Crystallizing a tough target

Dear Kavya, we encountered this issue for a protein complex at 95 mg/mL involving Human Serum Albumin (HSA) and a designed protein binder (Alphabody) as described in Pannecoucke et al. 2021 Sci Adv 7 (13), DOI:10.1126/sciadv.abe1682. The breakthrough in that case came from crystallization

Re: [ccp4bb] Crystallizing a tough target

Dear kavya There is precedent for this. Check the reference below. This protein was crystalized from solution at a concentration of 150 mg/ml. Keep concentrating, I guess ! Cell. Volume 98, Issue 4 , 20 August 1999, Pages 537-546

[ccp4bb] Crystallizing a tough target

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

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