<html><head><meta http-equiv="content-type" content="text/html; charset=utf-8"></head><body style="word-wrap: break-word; -webkit-nbsp-mode: space; -webkit-line-break: after-white-space;"><div style="color: rgb(0, 0, 0);"><font face="Arial">To whom it may concern, </font></div><div style="color: rgb(0, 0, 0);"><font face="Arial"><br></font></div><div><font face="Arial">I am currently working on segmenting a high resolution cryo-EM map (~4Å by the “gold standard FSC”) to phase crystallography data. After the segmentation, my colleague doing the phasing tells me that map appears more like 12Å resolution and I am worried I am doing something wrong during the process and somehow distorting my original data. I believe the 4Å of the original map based on structural features that I can clearly see. And the segmented density does not appear as defined. Therefore it appears that there has been some changes.</font></div><div style="color: rgb(0, 0, 0);"><font face="Arial"><br></font></div><div><font face="Arial">In order to begin the process, I did some manipulations before I started. This is an icosahedral virus map created through Auto3dem. I am only using an small quadrant of the original map, and since it was in pif format originally, I used proc3d to convert to mrc and invert the contrast to be compatible with segger. Before segmentation the map visually looked the same as the original pif map when both were displayed in chimera. </font></div><div style="color: rgb(0, 0, 0);"><font face="Arial"><br></font></div><div style="color: rgb(0, 0, 0);"><span style="font-family: Arial;">The procedure I followed after segmentation was: "</span><b style="font-family: Arial;">Save selected regions to .mrc file...</b><span style="font-family: Arial;"> save density map masked by the </span><a href="https://www.cgl.ucsf.edu/chimera/docs/UsersGuide/selection.html" style="font-family: Arial;">selected</a><span style="font-family: Arial;"> segmentation regions to an MRC file (map dimensions set to the minimal box containing the regions)”. </span><font face="Arial">I have made sure that the correct binning has been applied during segmentation. </font></div><div style="color: rgb(0, 0, 0);"><font face="Arial"><br></font></div><div style="color: rgb(0, 0, 0);"><font face="Arial">Any help would be much appreciated. Kristin </font></div><div style="color: rgb(0, 0, 0);"><font face="Arial"><br></font></div><div style="color: rgb(0, 0, 0);"><div><div><font face="Arial">-- </font></div><div><font face="Arial">Kristin N. Parent, Ph.D.<br>Assistant Professor<br>Dept. of Biochemistry and Molecular Biology<br>Michigan State University<br>603 Wilson Road<br>Room 519 Biochemistry Building<br>East Lansing, MI 48824</font></div><div><font face="Arial">Office phone: (517) 432-8434</font></div><div><font face="Arial"><a href="https://kparentlab.natsci.msu.edu">https://kparentlab.natsci.msu.edu</a>/</font></div><div style="font-size: 14px; font-family: Calibri, sans-serif;"><br></div></div></div><div style="color: rgb(0, 0, 0); font-size: 14px; font-family: Calibri, sans-serif;"><br></div><font face="Calibri,sans-serif" style="color: rgb(0, 0, 0); font-size: 14px;"> </font><br><div style="color: rgb(0, 0, 0); font-size: 14px; font-family: Calibri, sans-serif;"><br></div></body></html>