Hi Kristin, I think your approach to extract an approximate icosahedral asymmetric unit should work fine. I'm guessing all the trouble comes from the fact that the original pif map values are negative where the structure is located. Then when you use volume eraser it places zero values in the map where you erased and zero is a higher value than the density values for the structure. That messes up the display in Chimera. This is just a guess. Let me know if it is not right. You can see the range of density values in the volume dialog below the histogram and the current contour level density value. The solid cube display can be fixed by turning off "Cap high values at box faces" at the bottom of the Surface and Mesh Options panel of the volume dialog. But I suggest you make the density values positive so you don't run into further problems down the line. For example SSEHunter will probably fail if the density is inverted or highly shifted. Here's what to do about it. If the PIF map has inverted density, meaning that the most negative map values are actually the most dense parts of your structure then you should invert the map so that the most positive values are the densest. You can do that with Volume Filter (menu Tools / Volume Data), with Filter Type "Scale" choose the scale value to be -1. Another possibility is that your map values are simply shifted into the negative range (not inverted). You can use the above Volume Filter dialog to shift the structure density values into positive territory. You could for instance shift so the mean map value is zero. You could find the mean using Volume Mean, SD, RMS (menu Tools / Volume Data). Here's a video that demonstrates another way to extract an icosahedral asymmetric subunit. http://plato.cgl.ucsf.edu/chimera/videodoc/IcosWedge/index.html I don't think you should use exactly this because you need your subregion to extend beyond the asymmetric unit because the molecules will cross the asymmetric unit boundaries. A variant of the method shown in the video could allow you to extract one asymmetric unit and its neighbors (just color the markers for each of the asymmetric units you want the same color). Maybe that gives you a bigger subregion than you want. Tom Kristin Parent wrote:

Hello,

I was hoping you could help me out. I am trying to locate secondary structure elements in an individual subunit of an icosahedral virus from a cryo-reconstruction. I have the entire virion in pif format and would like to use the density from a single subunit using SSEHUnter and SSEBuilder. I am not sure how best to get the volume data as a useable mrc input for this tool.

I tried using the volume eraser to eliminate other areas in the map that are not the subunit I'd like. When I save the map and then the session, the subunit looks fine in the session, but if I open the subunit mrc file in a new session, I get what appears to be inverse density (the stuff I erased), and in the volume viewer, the density looks inverted. If I move the slide bar to the left, I end up with a solid cube. I am either doing something horribly wrong, or this is maybe not the best method for excising my subunit??? Please advise the best approach to creating a subunit mrc file that can be used by SSEHunter.

Thanks in advance! Kristin

Kristin Parent, Ph.D. University of California, San Diego Department of Chemistry and Biochemistry 9500 Gilman Drive, NSB 4104A, MC-0378 La Jolla, CA, 92093-0378 (858) 534-8038 _______________________________________________ Chimera-users mailing list Chimera-users@cgl.ucsf.edu http://plato.cgl.ucsf.edu/mailman/listinfo/chimera-users