[ccp4bb] Data Fitting for protein-ligand interaction.
Dear All, Firstly sorry for asking a non-crystallography question, but i want help in understanding the data analysis for fitting a protein-ligand binding data. Actually i have a protein which is a tetramer in solution and i have done its flourescence binding with a ligand. I am trying to fit the data to a 4-site binding model in scientist. But i donot have a correct model to fit in the data for identical or non-identical, co-operative or sequential binding. Can anyone help me in analysing the binding data. Any help will be highly appreciated. Thankyou !
[ccp4bb] Data Fitting for protein-ligand interaction.
Dear All I've been reading several mails that adress the problem of acetylated N-termini when refining peptide ligands with refmac. I managed to include LINKR records after running refmacs review restraints as suggested by Eleanor Dodson in one of the mails I found: LINKRC ACE I 0 N TRP I 1ACE_C-N But the records themself are obviously not sufficient to maintain the ACE linked to TRP during refinement and ACE moves away. Eleanor Dodson wrote about that topic: If you run [...] review restraints, it will detect and make a LINK entry for you Then you will need to use the GUI task - merge monomer library to combine your corrected MAL with the new LINK Run refmac again with XYZIN the output from review restraints task (that will include a LINK record) and it should/might! work... If I read that correctly, Review restraints should produce a LIBOUT that can be loaded as .cif in a later step for refinement? Such a file was not produced. I tried afterwards: 1) To regularize and safe an ACE-TRP monomer in JLIgand and load the .cif as LIBIN for refmac. The link is found, but ACE still moves away with the same messages: WARNING : link:ACE_C-N is found dist = 1.361 ideal_dist= 1.329 ch:II res: 1 TRP at:N .-Ia res: 0 ACE at:C . Even though XYZOUT still contains the LINKR records. 2) As I suspected, refmac needs except for the LINKR record a LIBIN that should be produced when running Review Restraints. As this .cif file is not produced, I sent the whole ligand to prodrug and safed the .cif from there. But that did not work out either. 3) I also tried to change the LINKR record into LINK I failed until now to tell refmac to maintain ACE linked to the TRP. I know this issues have been discussed before, but none of the suggestions helped yet. Any help would be highly appriciated.
Re: [ccp4bb] Data Fitting for protein-ligand interaction.
Hi Monica If protein is Homo-tetramer then one can expect the identical binding sites. I am also working on homo-dimeric protein which binds to DNA. I used PRISM to estimate the binding affinity through flourescence bindingmethod using “SATURATION and NON-LINEAR REGRESSION and ONE SITE binding Model’ considering protein concentration as dimer or monomer. Then I looked for the sensibility of the model by analyzing (comparing) best fit values (like SD, Bmax) of the parameters with reasonable certainty. Unlike ITC binding model fitting (gives good estimate of stoichiometry, cooperativity and also binding sites (one, two or sequential binding sites)), flourescence binding models do not give very good estimate of these parameters. Thus, based on protein you should assume the model which fits to the properties of the protein. Good luck Raj On Thu, Sep 12, 2013 at 12:59 PM, MONICA MITTAL monica.mitta...@gmail.comwrote: Dear All, Firstly sorry for asking a non-crystallography question, but i want help in understanding the data analysis for fitting a protein-ligand binding data. Actually i have a protein which is a tetramer in solution and i have done its flourescence binding with a ligand. I am trying to fit the data to a 4-site binding model in scientist. But i donot have a correct model to fit in the data for identical or non-identical, co-operative or sequential binding. Can anyone help me in analysing the binding data. Any help will be highly appreciated. Thankyou !
