On 9/19/2014 2:29 PM, Eleanor Dodson wrote: > hmm - crystallographically difficult. The usual way is to make a > dictionary file from the chemical information about the ligand. try to > build something obeying the chemical restraints into the density - > refine those coordinates and validate them. > Eleanor >
I would be a lot more cautious than this. It is good to remember that the density for a fully occupied, ordered ligand will be as strong and clear as that of the protein in the active site. The fact that you have done some refinement but are still not sure if your ligand, or anything for that matter, has bound means that at best your crystal has problems. You have two questions. Did anything bind during your soak? Did the ligand of interest bind? The first you can attack fairly unambiguously with an Fo(holo)-Fo(apo) map. I presume you have an isomorphous apo data set since you are performing a soaking experiment. A clear set of density that stands out above the background in the Fo-Fo map tells you that something new is in your crystal. Figuring out what it is is another matter and is very difficult if the density is weak. Eleanor is recommending building and validating. Unfortunately none of the validation tools we have have hard cutoffs of what is acceptable and what is not. With years of experience one can come to a conclusion, but someone starting out doesn't really know if those tests are definitive. Remember you are not trying to decide if your ligand bound, but if the thing that bound is your ligand. There are many other things that could happen. Maybe your ligand bound. Maybe something else in your solution bound. Maybe the chemical in your solution wasn't what you thought it was. Maybe it was, but has been chemically transformed since then and is now something else. Maybe this particular crystal had something in it all along that your apo crystal didn't. This isn't a matter of seeing IF you can build your ligand into the density and get away with it. You have to also say that you CAN'T build anything (reasonable) in that density OTHER THAN your ligand. With weak and fuzzy density this is very hard to do. If you performed a soak and got something to bind, maybe you could soak longer or at higher concentration and get a more definitive map. If you have not solved the structure of a protein:ligand complex before I suggest you check out other models in the PDB with good, strong density and see what a full power ligand looks like in a map. Only settle for weak density if there is no alternative and never settle for ambiguous density. Dale Tronrud > > On 19 September 2014 22:06, ansuman biswas <[email protected] > <mailto:[email protected]>> wrote: > > Dear All, > > I have collected a diffraction dataset from a crystal soaked in a > solution containing the ligand of interest. > After refining a few cycles, I can see some density in the active > site pocket, but not so clear to model the ligand unambiguously. > > Is any tool available to validate whether the ligand is actually > there or not ? > The Twilight server appears to be for PDB files that have already > been deposited. > > thanks and regards, > Ansuman Biswas, > dept. of Physics, > Indian Institute of science > >
