I'm a fairly inexperienced crystallographer, but I have worked on a few cases very recently (from separate protein systems) in which the ligand was not "clear" in the electron density until quite a long way through refinement: in the latest case, it wasn't possible to be too confident of the ligand's presence (rather than, say, water) until the R/Rfree were approx 21/26% respectively, and only after several more rounds of refinement were we confident of the orientation (R/Rfree 18/23%).
As I said at the top, I'm fairly inexperienced, so I'm not yet sure whether this is an extreme case, but I think it's definitely worth doing molecular replacement and refining as much as possible before "giving up" on a dataset! The change in electron density quality - especially in the region of the ligand - steadily improved throughout refinement. One thing also worth remembering is that the occupancy of the ligand is not necessarily 1. I was nearly put off a dataset completely because of this, until a considerably more experienced colleague pointed out that it's probably not in every single binding site - after playing around with the occupancy (from 1.0 to 0.85), the model suddenly became very reasonable, which was nice :) Of course, the caveat is not to over-refine... Isaac -----Original Message----- From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of Palm Sent: 29 May 2007 21:25 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] How to determine ligand binding from diffraction pattern? I would like to expand on the question and answer below and compare your experiences: Looking for ligands in many different soaks / cocrystals of your protein of interest, you still should do molecular replacement and a bit of refinement. I agree with Steve, but how much refinement is necessary and enough? We have a specific case with a 24 kDa protein crystallizing in P6522 with resolution of 2.5 - 3 A, which should be comparable to most cases. The ligands have 10 - 20 non-hydrogen atoms (most of the time we don't know, we are actually screening for them). How far should we refine to see if we have only water molecules or a ligand bound - to an Rfree of 0.45 or 0.40 or 0.35? greetings Gottfried Dear all, Is there a simple way to determine whether ligand is bound or not by comparing the diffraction patterns between ligand-free (structure known) and ligand-soaked protein crystals? I would like to solve the ligand bound protein structure, but before I do so, I have to find out if the ligand is actually bound. Thank you very much! Best, Joe Having done this a few hundred times, I would strongly suggest that you just collect the data and solve the structure. Since you already have the apo structure solved, then it really isn't that much work to do an MR solution on the complex. Be aware that quite frequently there is enough non-isomorphism to necessitate partial refinement of the "complex" structure before recognizable density will appear for the ligand. The definitive answer can only be obtained with a full data set, so go for it. Good luck- Steve No virus found in this outgoing message. Checked by AVG Free Edition. Version: 7.5.472 / Virus Database: 269.8.1/822 - Release Date: 28/05/2007 11:40
