Try GraphEnt but you might have to recompile it for your size of proteins. That has helped me several times to see something that was barely there. Here's the link for the 'click-generation': http://utopia.duth.gr/~glykos/graphent.html
Jürgen ...................... Jürgen Bosch Johns Hopkins University Bloomberg School of Public Health Department of Biochemistry & Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Office: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-2926 http://lupo.jhsph.edu On Oct 4, 2012, at 3:17 PM, Israel Sanchez wrote: Hello everyone, I would like to share my experience with one dataset and request some advice on which is the best way to prove a conformational change seen in a density map. The first issue arose when we were looking for an extra ribosomal factor added to a crystalized ribosome. After careful data collection and refinement (I/sigma last shell 1.2, 3.1A and CC1/2 around 22%) the sigma-A-weighted maps 2mFo-DFc and Fo-Fc does not show any clear difference density that we could interpret as the expected factor. Interestingly, a computed map with coefficients 3mFo-2DFc started to show some features that clearly could be explained as a fragment of the factor. The density improved even more with a B-sharpened map. We have seen this behavior before and I was wondering if someone else is using this kind of maps and may could explain the reason behind this density improvement. Is it a crazy idea to go even higher like 4mFo-3DFc? The second query has to do with which is the best way to prove that a conformational change is present in an specific residue (in this case and RNA base) in your structure. To my knowledge, a classic omit map with simulated annealing would do the job regarding removing the model bias. Actually, I found an interesting alternative in PHENIX called a Kick map, were a series of maps computed from a ramdoinised set of models yields a averaged map ideally free from model bias. Does anyone has a preference for any of those schemes? Are there more alternative to prove a conformational change in a model phased with a molecular replacement solution? Thank you very much in advance. -- Israel Sanchez Fernandez PhD Ramakrishnan-lab MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH, UK
