<div dir="ltr"><div><div><div><div><div>Hi Reza,<br><br></div>Second question first - not much (I can't detect any), assuming same scope and mag (which is relieving for multiple reasons). However, I don't know how close the pixel size I obtain by real space correlation with a crystal structure is to the "true" pixel size, and whether it is wrong in any systematic way.<br><br></div>Re the other questions, I have not compared rigorously (I only refined against the map with the correct pixel size), but I undertook steps to correct it when I noticed during initial building in Coot that known (solved) domains were not quite fitting the density as well as expected. I would expect some consequences from this, particularly in terms of clash analysis and the use of reference restraints (not sure about electrostatics etc).<br><br></div>Where it does make a big difference is in comparing structures of the same or related proteins solved by different groups - if the pixel size varies by even 1-2%, this really messes up structural superposition for large structures (if the structure is 300Å, this could be a 6 Å difference in size). In my experience many groups don't seem to do this - there are quite a lot of recent maps in the EMDB in which the nominal pixel size has been taken as correct, and calibration with a crystal structure shows that it is a ways off (this also affects the estimated resolution, sometimes substantially).<br><br></div>Cheers,<br></div>Oli<br><div><div><div><div><div><div><div><div class="gmail_extra"><br><div class="gmail_quote">On Mon, Feb 8, 2016 at 11:38 AM, Reza Khayat <span dir="ltr"><<a href="mailto:rkhayat@ccny.cuny.edu" target="_blank">rkhayat@ccny.cuny.edu</a>></span> wrote:<br><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
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<p>Hi Oliver,<br>
</p>
<p><br>
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<p>Out of curiosity:</p>
<p> 1. what kind of R-factors and CC-values do you get when refining against the two different pixel size? <br>
</p>
<p> 2. how different are your refined pixel sizes from one reconstruction to another?<br>
</p>
<p> 3. how much of an affect does the wrong pixel size have on your downstream structure analysis (e.g. BDA, ASA, electrostatic...)? </p>
<p><br>
</p>
<p>Best wishes,<br>
Reza<br>
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</p>
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Reza Khayat, PhD
<div>Assistant Professor </div>
<div>City College of New York</div>
<div>Department of Chemistry</div>
<div>New York, NY 10031</div>
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<div dir="ltr"><font style="font-size:11pt" face="Calibri, sans-serif" color="#000000"><b>From:</b> <a href="mailto:phenixbb-bounces@phenix-online.org" target="_blank">phenixbb-bounces@phenix-online.org</a> <<a href="mailto:phenixbb-bounces@phenix-online.org" target="_blank">phenixbb-bounces@phenix-online.org</a>> on behalf of Oliver Clarke <<a href="mailto:olibclarke@gmail.com" target="_blank">olibclarke@gmail.com</a>><br>
<b>Sent:</b> Monday, February 8, 2016 4:28 AM<br>
<b>To:</b> <a href="mailto:phenixbb@phenix-online.org" target="_blank">phenixbb@phenix-online.org</a><br>
<b>Subject:</b> [phenixbb] Refine pixel size of map (for EM data)?</font>
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<div>Hello,<br>
<br>
</div>
I wonder whether it would be possible to add an option for phenix.real_space_refine to allow refinement of the pixel size of the map (or the unit cell dimensions - just an overall size scale factor), and write out the altered map at the end of refinement.<br>
<br>
Although we try to calibrate this as best as we are able at the time of data collection, it is never perfect - for example, in one case I have dealt with, our nominal pixel size out of the scope is 1.19 Å, but the pixel size calibrated based on a crystal structure
of a fragment of the protein is 1.25 Å. This is not a huge difference, but it is sufficient I think to have a substantial impact on refinement, particularly as regards clash assessment and H-bond/sec struc restraints.
<br>
<br>
In cases where one does not have a solved crystal structure to use for calibration, perhaps refining the pixel size in conjunction with the geometry might be of some use?<br>
<br>
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<div>Cheers,<br>
</div>
<div>Oli<br>
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