Have you got two very similar datasets (isomorphous) inbetween which the mentioned difference should be pronounced? You could try a cross-crystal difference map as well.
Jan On Fri, Oct 5, 2012 at 8:40 AM, <[email protected]> wrote: > ** > Dear Israel, > > I wonder why you do not see anything in your Fo-Fc (mFo-DFc?) maps, since > the limit for a (n+1) * mFo - n * DFc map, when n approaches inifinity, is > the mFo-DFc difference map. There is nothing wrong in going further like > with 4mFo-3DFc maps, but you should bear in mind that they are becoming > more and more difference map-like and less and less regular maps. > > What I think is happening is that since you added your ribosomal factor to > preformed crystals, you have incomplete occupancy, say the factor bound > only to 50% or less of the ribosomes. This means that you have to go down > in contour level from the 3 sigma which is the default for difference maps > to maybe 1.5 or even 1 sigma. If the factor is bound indeed at lower level > you will see relatively nice density, if nothing is there, you will see > only noise. > > To prove a conformational change at a single residue, I would just leave > it out and refine. Since the refinement program has no reason to bias > either conformation, the result should be unbiased. However, in the case of > partial occupancy, the resulting map will be a superposition of both > conformations. Also to reduce model bias, you might want to try the Buster > program. > > Best regards, > Herman > > ------------------------------ > *From:* CCP4 bulletin board [mailto:[email protected]] *On Behalf Of > *Israel > Sanchez > *Sent:* Thursday, October 04, 2012 9:17 PM > *To:* [email protected] > *Subject:* [ccp4bb] On maps and doubts > > 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 > > > -- Jan Dohnalek, Ph.D Institute of Macromolecular Chemistry Academy of Sciences of the Czech Republic Heyrovskeho nam. 2 16206 Praha 6 Czech Republic Tel: +420 296 809 340 Fax: +420 296 809 410
