Hi, First, there is nothing magical about contouring a map at 1 rms. The standard deviation of the electron density values really has no relationship to the rms of those values, and appears to generally be much smaller. This is discussed quite brilliantly in the recent paper http://www.ncbi.nlm.nih.gov/pubmed/24363322
If you have a ligand with low occupancy you expect you will have to "dial down" the contour level to see it. The question isn't how low can you go but does your model fit ALL the available data and is there any other model that will also fit those data. Even if a ligand has low occupancy it still must have good bond lengths and angles and must make reasonable interactions with the rest of the molecule. One of your observations is that full occupancy cracks the crystal. It would be good if your model explains this observation as well. If your ligand is present 60% of the time, what is there the other 40%? Usually when there is a partially occupied ligand there is water present the rest of the time. The apparent superposition of the ligand and the water will result in some density that is strong. Those strong bits will give clues about the minor conformation. The low occupancy water model must also make sense in terms of hydrogen bonds and bad contacts. Remember, if you are looking at lower than usual rms contours in the 2Fo-Fc style map you must evaluate your refined model by looking at lower contour levels in your Fo-Fc style map. You can't give your model a free ride by excusing a weak density map but then blowing off weak difference peaks. You must be very careful to consider alternative models and to accepts that sometimes you just can't figure these things out. Just because the density is weak does not mean that you can give it a pass for not "fitting" the model. The model has to fit the density, and fit it better than any other model. You must also make clear to your readers what the occupancy of your ligand is and the quality of the maps that lead you to this conclusion. Dale Tronrud P.S. I have had great experiences with the maps produced by Buster for looking at weak ligand density. I have also published a model with a 0.35 occupancy ligand although the resolution there was 1.3 A. On 03/19/2014 07:39 AM, Amit Kumar wrote:
Hello, My protein is 26 kDa and the resolution of the data is 1.90 angs. My ligand is 174 Daltons. and it was soaked into the crystal. Ligand is colored and the crystal after soaking takes up intense color. However if we soak more than optimum, the color deepens in intensity but the crystal diffracts no more. So perhaps the ligand's occupancy can not be the 1.00. After model building I see ligand density, starting to appear at 0.7 sigma and clear at 0.5-0.6 sigma, close to the protein residue where it "should" bind. Occupancy is ~0.6 after the refinement and B factors for the atoms of the ligand range from 30-80. Questions I have (1) What is the acceptable sigma level for very small ligands for peer review/publication? (2) I did refinement by Refmac and by Phenix refine, separately. The map quality for the ligand is better after the refmac refinement than after the Phenix refinement. Why is such a difference and which one should I trust? I used "mostly" default parameters for both (Phenix and Refmac) before the refinement. Thanks for your time. Amit
