Your holo structure has a Ca++ and three water molecules that have not been built into your low resolution apo map. These atoms are not expected to be resolved at 3 A resolution, so I would expect them to appear as a large, misshapened, blob. Your screenshot only shows one contour level. It is quite possible that the highest density value is not at the center of the blob.
You might have a lower occupancy Ca++ atom at the site and the image is confused by the low resolution. Remember, even if the concentration of Ca++ is lower in this mother liquor any Ca++ that binds will bind exactly as it does in the fully occupied case. A weakly binding Ca++ site will not bind before the strongly binding site. I would first look to see what your holo map looks like when it's resolution is truncated to 3 A. This will give you a sense of what a Ca++ binding in this site would look like. You could try refining a model with the Ca++ and water molecules, with lower occupancy, and see what the residual difference map looks like. You will, of course, have to have strong restraints on the geometry to hold this model together at 3 A resolution, but fortunately you have a higher resolution model to base these restraints on. The PDB file is a statement of your belief of what is in the crystal. Don't waste your time refining models that don't make chemical sense. An ion floating in space with no ligands is not a reasonable model so even if it "fits" the density it can't be correct. There a multiple ways of justifying the model of a crystal and others on the list will likely have different ideas for the criteria that should be used. My belief is that you know the holo model and the most likely outcome of your Ca++ extraction experiment (in a Bayesian prior sense) is a lower occupancy binding of the Ca++ and its water molecules. If you build and refine that model and the difference map is acceptable you can say that this model is consistent with your experiment. If there is residual density then you can conclude that something is replacing the Ca++, but untangling superimposed, partial occupancy, models at 3.1 A resolution is extremely difficult. I think all you will be able to say is that "something replaces the Ca++ but it cannot be identified". Not everything can be identified in a 3 A map. Not everything can be identified in a 1 A map. Your job is to say "these parts I understand and these parts I don't". Dale Tronrud On 05/15/12 07:51, RHYS GRINTER wrote: > Dear Community, > > As I'm a relatively new to protein crystallography this might turn out to be > an obvious question, however. > > I'm working on the structure of a enzyme requiring Ca2+ for activity and with > calcium coordinated in the active site by Asp and 2x backbone carbonyl > groups, in a crystal structure with Ca in the crystallisation conditions > (http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_TD_15A.jpg). > When Ca is omitted from the crystallizing conditions and a divalent chelator > (EGTA) is added the crystals are of significantly lower resolution (3.13A). > Refinement of this data reveals density for a molecule coordinated by the Ca > coordinating Asp and backbone, however this density is significantly further > away (3.4-3.8A) too far away for water or a strongly coordinated divalent > cation(http://i1058.photobucket.com/albums/t401/__Rhys__/MDC_EGTA_315.jpg). > The density is also much weaker than for Ca in the previous model > disappearing at 3.5 sigma. > > The crystallisation conditions for the Ca free condition is: > > 0.1M Tris/Bicine buffer [pH 8.5] > 8% PEG 8000 > 30% Ethylene Glycol > 1mM EGTA > > The protein was purified by nickel affinity/SEC and dialysed into: > 20mM NaCl > 20mM Tris [pH 8.0] > > > A colleague suggested that sulphate or phosphate could fit at these > distances, but these ions have not been added at any stage of the > crystallisation process. > > > Could anyone give me some insight into what this density might represent? > > Thanks in advance, > > Rhys Grinter > PhD Candidate > University of Glasgow
