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Refinement: Difference between revisions
→Refining low resolution structures
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If you are really desperate, another option could be to use harmonic restraints in [[CNS]] to keep your backbone fairly fixed in parts of the map where you believe the secondary structure is correct (most likely alpha-helices). You could also fix main-chain elements completely (in any refinement program), but it is definitely preferable to leave some room for change in the xyz positions, and harmonic restraints are a nice way of doing exactly that. | If you are really desperate, another option could be to use harmonic restraints in [[CNS]] to keep your backbone fairly fixed in parts of the map where you believe the secondary structure is correct (most likely alpha-helices). You could also fix main-chain elements completely (in any refinement program), but it is definitely preferable to leave some room for change in the xyz positions, and harmonic restraints are a nice way of doing exactly that. | ||
=== Bulk solvent correction produces difference density === | |||
Sometimes people observe strong residual difference density in a cavity of the protein. E.g. there was a paper by Brian Matthews' group (Marcus D. Collins, Michael L. Quillin, Gerhard Hummer, Brian W. Matthews, Sol M. Gruner, Structural Rigidity of a Large Cavity-containing Protein Revealed by High-pressure Crystallography, Journal of Molecular Biology, Volume 367, Issue 3, 30 March 2007, Pages 752-763, [http://dx.doi.org/10.1016/j.jmb.2006.12.021]) on a high pressure form of lysozyme where they found a large hydrophobic void. Bulk water could only be compelled to enter the void by application of very high external pressure. | |||
Bulk solvent mask artifacts can only occur at narrow channels, where the mask radius is too big to define the channel as belonging to the bulk solvent region, leaving it "empty" and thus resulting in ''positive'' difference density. | |||
The following advice is specific for [[ccp4dev:Refinement_with_Refmac5|Refmac]]: Changing from simple scaling to Babinet scaling is an important check to exclude mask bulk solvent artifacts, but there, you have to uncheck the "calculate contribution from the solvent region", because this is done by the Babinet scaling, already. | |||
For [[Phenix|phenix.refine]], the bulk solvent mask may be varied - see [http://www.phenix-online.org/documentation/refinement.htm#anch52]. | |||
In the case of ''negative'' difference density in a big hydrophobic cavity, one possible reason for a negative difference density are underestimated magnitudes of |Fobs| at very low resolution, either because they are weakened by the beam-stop (half-)shadow, or because they are overloads that have been poorly extrapolated. A simple check for wrongly determined low-resolution |Fobs| is to cut your low resolution data during refinement at a somewhat higher resolution, say 20 A instead of 80 A, and see whether the negative difference density disappears. If, yes, you should check your data processing again. | |||
The other possibility of course is that the data is good, that this is an accurate experimental result and there really is a void, or at least a cavity where the mean bulk density is lower than in bulk water. One way to test the void theory would be to fill the cavity with O atoms of zero (or very small, say 0.01) occupancy. Hopefully (!) that will prevent Refmac filling the cavity with bulk solvent. One could then try giving these O atoms large B factors, say 200, to smear them out, and then increase the occupancies to titrate the actual bulk density. | |||