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

Dear Patrick, Thank you for the suggestions and for the detailed the statistics!! Regards Kavya On 2024-02-06 23:06, Patrick Shaw Stewart wrote: > Hi Kavya > > 1. Make a seed stock from the globules or anything else that you think might > be crystalline, and recreen. In other words,

Re: [ccp4bb] Crystallizing a tough target

Hi Kavya 1. Make a seed stock from the globules or anything else that you think might be crystalline, and recreen. In other words, you should add your seed stock to *random screens* (not optimization experiments). There could be many conditions that are in the metastable zone of the phase

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

From: CCP4 bulletin board on behalf of Jon Cooper <488a26d62010-dmarc-requ...@jiscmail.ac.uk> Sent: Monday, February 5, 2024 5:32 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [External] Re: [ccp4bb] Crystallizing a tough target Limited proteolytic might help, too. https://www.natu

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] 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