[ccp4bb] ITC with heterogeneous protein

Dear All, This is an off-topic question. I have protein solution of heterogeneous (contains both monomer and dimer). I want to perform ITC with this protein. I doubt whether this heterogeneity will interfere the binding study. Any advice please. Thank you Sajid

Re: [ccp4bb] ITC with heterogeneous protein

Dear Sajid, one first problem in your study is how-to adress if the deltaH mesured is caused by the ligand interaction, or by the modification of dimer-monomer equilibrium. You have to well caracterise your system dimer-monomer. One other problem is about the accessibility of the interaction

Re: [ccp4bb] ITC with heterogeneous protein

Dear Sajid If the binding site of your ligand is remote from the dimerization interface, it should normally not be a problem. You will bind two ligands for a dimer and one ligand for a monomer and you should be able to fit the isotherm with one unique site even in presence of a mix of monomers

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Re: [ccp4bb] ITC with heterogeneous protein

Hi Sajid, *Assuming* you have one site per monomer (rather than, say, one site per dimer), and *assuming* each binding event is completely independent ( I.e no co-operativity), you might just get away with running the experiment with the heterogeneous material. However, you might not be able to

Re: [ccp4bb] ITC with heterogeneous protein

I have a similar case, where in there are multiple binding sites on the protein for the ligand and ligand induces dimerization.So it is not helpful even if I separate the monomer and dimer. If I titrate the dimer with ligand, the stoichiometry will completely change? Any suggestions will be

Re: [ccp4bb] ITC with heterogeneous protein

it depend of what you expected as information : In this case your measure is resulting from ligand binding AND dimerization (except if all the protein is already dimerized). I am not sur to understand, do you know how many binding sites exists on one monomer ? You should be able to determine the

[ccp4bb] MBP-tag

Sorry for the off-topic, but I need some help. I have a protein with a HisTag. I need to deglycosylate it prior to crystallisation, as the glycosylation trees interfere in the process. As I didn't want to change the buffer the protein is in, I use the enzyme EndoHf (which has an MBP-tag), and

[ccp4bb] Negative electron density in the Fo-Fc map at the binding site

Dear all, I am screening a small library of ligands against my protein crystals. Following a soaking with different ligands, I collect datasets to 1.9A resolution and refine them against an empty model without any problem. What is the meaning of a rather large negative electron density in

Re: [ccp4bb] Translational NCS and molecular replacement.

Dear community, Thanks for your valuable suggestions. Best regards, Sudipta. On Thu, Jul 17, 2014 at 8:04 AM, Eleanor Dodson eleanor.dod...@york.ac.uk wrote: No need to reindex - just do this to change the space group in the scala header. mtzutils hklin1 scala.mtz hklout scala-P22121.mtz

[ccp4bb] Electron scattering factors for CNS

Dear CCP4BB members, I wonder if there is a CNS library file for electron scattering factors. Many thanks! Minglei Minglei Zhao, Ph.D. Stanford University, School of Medicine James H. Clark Center 318 Campus Drive, Room E300 Stanford, CA

Re: [ccp4bb] Negative electron density in the Fo-Fc map at the binding site

Hi Armando, can be many reasons, a few for instance: - are you sure the ligand you modeled in is the one that is actually there? - is the positive density around or overlaps with ligand atoms you places? Then that would means ligand parameters are underrefined (need more refinement) or not

Re: [ccp4bb] Electron scattering factors for CNS

Hi Minglei, I can't remember if CNS has this, but I it is available in Phenix. Pavel On Fri, Jul 18, 2014 at 2:22 PM, Minglei Zhao mlz...@mbi.ucla.edu wrote: Dear CCP4BB members, I wonder if there is a CNS library file for electron scattering factors. Many thanks! Minglei

Re: [ccp4bb] ITC with heterogeneous protein

If the following is not deleterious to your protein and its function you could introduce mutations that prevent dimerization. ~Jeff I have a similar case, where in there are multiple binding sites on the protein for the ligand and ligand induces dimerization.So it is not helpful even if I