<div dir="ltr"><div class="gmail_quote"><div dir="ltr"><div class="gmail_extra"><div class="im">On Fri, Oct 11, 2013 at 6:19 PM, Ryan Spencer <span dir="ltr"><<a href="mailto:rspencer@uci.edu" target="_blank">rspencer@uci.edu</a>></span> wrote:<br>
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<blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"> For high resolution structures, below 1.5 Å, is there a set of rules<br>
for best practices for refinement?<br></blockquote><div><br></div></div><div>Not really rules that I'm aware of... just general guidelines, and not well-codified. I've wanted for a long time for us to write some sort of extensive how-to across all resolution ranges, but like everything else, this has to compete with writing code and "real" papers. As Pavel mentioned, these resolution limits are somewhat subjective depending on completeness, how aggressively you process the data, etc.</div>
<div><br></div><div>But, a few basic suggestions:</div><div><br></div><div>- you will probably not want to use real-space refinement after the first couple of rounds</div><div>- start parameterizing conservatively, then get more aggressive as you near the end of refinement (if nothing else, it's faster this way)</div>
<div>- don't use any restraints other than the default geometry restraints plus whatever custom bonds are required by the chemistry (e.g. prosthetic groups)</div><div>- use hydrogens by the end of refinement, but riding only until you're at subatomic resolution</div>
<div>- the automatic weighting is probably going to work great; weight optimization might help for the final round, but probably not necessary</div><div>- below 1.5Å, try making non-water/non-hydrogen anisotropic (and compare to purely isotropic); below 1.2Å, you can start thinking about making all non-H/D anisotropic. (Also note that heavier atoms can become anisotropic even earlier, so in your case, refining individual anisotropic B-factors for iodine and TLS for everything else would be a reasonable alternative.)</div>
<div>- you should probably be able to get rid of all geometry outliers, including clashes - but you may see real (correct) Ramachandran or rotamer outliers at this point</div><div>- you're inevitably going to see more blobs in the difference map that you can't explain than usual. don't sweat it.</div>
<div>- you should definitely see a fair number of alternate conformations - I don't know what is expected statistically but I'd say at least 10% of protein residues will have them</div><div>- pay attention to waters with close contacts to oxygen - these may be sodium (or other) ions</div>
<div><br></div><div>I've attached a few slides that give *very approximate* suggestions - basically just reframing what Pavel and others have said over the years. Although some of these are pretty clear-cut (e.g. you absolutely do not want to use a reference model), you will always need to experiment somewhat to find out what is best for *your* model and data.</div>
<div><br></div><div>I think based on your R-factors and statistics that you're headed in the right direction. I'd try to get the clashscore as close to zero as possible, though.</div><div class="im"><div><br></div>
<blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
I've been toying with different parameters and found that the<br>
weighting didn't make a huge difference, which is probably expected, with a<br>
1.3 Å dataset. Do stereochemical/ADP weights have any effect?<br></blockquote><div><br></div></div><div>Do you mean weight optimization? I wouldn't expect it to make much difference.</div><div class="im"><div> </div>
<blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
For the particular data set I'm working with, I'm refining directly<br>
against anomalous data, more specifically against 12 Iodines. Refining the<br>
f' and f" has worked well, generating a beautiful density map.<br></blockquote><div><br></div></div><div>Good idea. Also consider looking at the anomalous residual map (map_type=anom_residual), which may show weaker anomalous scatterers like chloride or sulfur. (It will be flattened around the iodines if you refine their f'' values.)</div>
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<div><br></div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
Refining against the model with hydrogens increased the Rfree/Rwork<br>
values by about 2 points. From what I've read previously I thought refining<br>
hydrogens in a non-"riding" model was only valid below ~1.0 Å.<br></blockquote><div><br></div></div><div>I'm not sure what you mean here. Did you use the riding model and it increased R-free? Or only when you let them refine individually? The default is now to switch to individual only at 0.9Å or less.</div>
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<div> </div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">by a few points. For water, I assume that at this resolution there is a<br>
combination of anisotropic (big banana looking density) and isotropic waters<br>
and that one cannot simply rely entirely on the water_update option.<br></blockquote><div><br></div></div><div>Yes, at some point you will need to complete the waters yourself. Those bananas may not be simply anisotropic waters, but split sites. You may see other small ligands too.</div>
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<div><br></div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex"> The twin law has worked great, usually dropping Rfree/Rwork values<br>
by 5 points or so.<br></blockquote><div><br></div></div><div>Just remember to be careful when interpreting the effect of twin law application - I think in this case it's probably quite reasonable, but at lower resolution and with a worse model it can be extremely deceptive.</div>
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<div><br></div><blockquote class="gmail_quote" style="margin:0 0 0 .8ex;border-left:1px #ccc solid;padding-left:1ex">
I apologize if any of this is common knowledge and shows my relative<br>
inexperience when it comes to best practices for crystal refinement.<br></blockquote><div><br></div></div><div style>No need to apologize - it is all oral tradition anyways. It is much better to ask questions now than to do something blindly and regret it later.</div>
<div style><br></div><div>-Nat</div>
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