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Samples can be prepared using, for example, <a href="https://www.nature.com/nprot/journal/v3/n2/full/nprot.2007.474.html">a protocol by Grassucci <em>et al</em>.</a>, and EM images obtained.</p> <p>What is important to note, is that the re-constructed 3D image is generated using many thousands of macromolecules (particles). In the figure below, for example, a total of 29,926 particles from 616 CCD frames (4k × 4k frames from a charge-coupled device camera) were used to produce the 4.3 Å resolution reconstruction shown in panel a. (taken from<a href="https://www.nature.com/nature/journal/v463/n7279/full/nature08701.html"> Zhang <em>et al</em>., 2010</a>)</p> <p><a href="https://www.nature.com/nature/journal/v463/n7279/fig_tab/nature08701_F1.html"><img class="alignnone" src="https://www.nature.com/nature/journal/v463/n7279/images/nature08701-f1.2.jpg" alt="" width="480" height="457" /></a></p> <p>Image reconstruction is therefore a significant part of the process of generating meaningful data, and it is important that the averaging process doesn’t overfit the data (my understanding of this is that overfitting relates to finding patterns in the parts of the data that are really just noise; and that this over-interpretation of the data would lead to an over-estimate of the true resolution of the image).</p> <p><a href="https://www.nature.com/nprot/journal/v3/n6/full/nprot.2008.62.html">One of our protocols for image analysis</a> involves the use of the software <a href="https://xmipp.cnb.csic.es/twiki/bin/view/Xmipp/WebHome">XMIPP</a>. In a recent letter to <em>Nature Methods</em>, Scheres and Chen introduce a script that can be used on top of the conventional projection-matching protocol in the XMIPP package to help prevent data overfitting.</p> <p>The article can be accessed here:</p> <p><a href="https://www.nature.com/nmeth/journal/v9/n9/full/nmeth.2115.html">Prevention of overfitting in cryo-EM structure determination</a></p> <p>The script is included in the Supplementary Information (should be accessible without a site licence):</p> <p><a href="https://www.nature.com/nmeth/journal/v9/n9/extref/nmeth.2115-S1.pdf">https://www.nature.com/nmeth/journal/v9/n9/extref/nmeth.2115-S1.pdf</a></p> <p>…………………………………………………</p> <p><strong>Nature Protocols relating to CryoEM of macromolecules:</strong></p> <p><a href="https://www.nature.com/nprot/journal/v2/n12/full/nprot.2007.452.html">Preparation of macromolecular complexes for cryo-electron microscopy</a><br /> Robert A Grassucci, Derek J Taylor & Joachim Frank</p> <p><a href="https://www.nature.com/nprot/journal/v3/n2/full/nprot.2007.474.html">Visualization of macromolecular complexes using cryo-electron microscopy</a> with FEI Tecnai transmission electron microscopes<br /> Robert A Grassucci, Derek Taylor & Joachim Frank</p> <p><a href="https://www.nature.com/nprot/journal/v3/n12/full/nprot.2008.156.html">SPIDER image processing for single-particle reconstruction of biological macromolecules from electron micrographs</a><br /> Tanvir R Shaikh, Haixiao Gao, William T Baxter, Francisco J Asturias, Nicolas Boisset, Ardean Leith & Joachim Frank</p> <p><a href="https://www.nature.com/nprot/journal/v3/n6/full/nprot.2008.62.html">Image processing for electron microscopy single-particle analysis using XMIPP</a><br /> Sjors H W Scheres, Rafael Núñez-Ramírez, Carlos O S Sorzano, José María Carazo & Roberto Marabini</p> <p><a href="https://www.nature.com/nprot/journal/v5/n10/full/nprot.2010.126.html">Cryo-EM of macromolecular assemblies at near-atomic resolution</a><br /> Matthew L Baker, Junjie Zhang,Steven J Ludtke & Wah Chiu</p> <p> </p> <p> </p> <p> </p> <p> </p> </div> </section> <div class="wpn-entry-meta"> <aside> <div class="article-tools cleared"> <h2 class="hidden">Article tools</h2> <ul id="wpn-share-post-621" class="wpn-entry-user-actions"> <li class="wpn-entry-comment-link"><a class="comments-link" href="https://blogs.nature.com/stepwise/2012/10/09/preventing-overfitting-during-the-reconstruction-of-macromolecule-images-from-cryoem-data#respond" title="Comment on Preventing overfitting during the reconstruction of macromolecule images from CryoEM data">add a comment</a></li> <li class="sendtofriend"><a href="https://blogs.nature.com/stepwise/tell-a-friend?id=621">Email</a></li> <li class="print"><a id="print-link" class="track" href="javascript:window.print();">Print</a></li> <li class="wpn-share-bookmark"> <h3 class="toggle-bookmarking-links">Share/bookmark</h3> <div id="bookmarking-links" class="bookmarking-popup"> 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href="https://blogs.nature.com/">nature.com blogs home</a> </div> <div id="wpn-npg-recent-comments-widget-2" class="wpn-widget wpn-comment-text wpn-npg-recent-comments-widget wpn- wpn-cache-miss"><h2 class="box-heading">Recent comments on this blog</h2><ul> <li><div><q>Congrats for 10th anniversary :) And thanks for update about your protocol extension.</q> ... <a href="https://blogs.nature.com/stepwise/2016/02/04/protocolextension#comment-73" class="more-link">Read more</a></div><span class="comment-author vcard">-- <cite class="fn">Raza Bajwa</cite></span><h2 class="wpn-post-title"><a href="https://blogs.nature.com/stepwise/2016/02/04/protocolextension">Introducing a third article type – the Protocol Extension</a></h2></li> <li><div><q>I have been very thankful for such wonderful post. Its really contain good information, which is knowledgeable. As we are…</q> ... <a href="https://blogs.nature.com/stepwise/2011/05/31/membrane-protein-protocols#comment-71" class="more-link">Read more</a></div><span class="comment-author vcard">-- <cite class="fn">Allen Christopher</cite></span><h2 class="wpn-post-title"><a href="https://blogs.nature.com/stepwise/2011/05/31/membrane-protein-protocols">Membrane protein protocols</a></h2></li> <li><div><q>verry interesting and difficult stuff.</q> ... <a href="https://blogs.nature.com/stepwise/2012/09/07/proteomics-and-protease#comment-53" class="more-link">Read more</a></div><span class="comment-author vcard">-- <cite class="fn">Hans Vlaming</cite></span><h2 class="wpn-post-title"><a href="https://blogs.nature.com/stepwise/2012/09/07/proteomics-and-protease">Label-free proteomics – the protease matters</a></h2></li> </ul></div><div id="wpn-rss-4" class="wpn-widget wpn-widget-rss wpn-"><h2 class="box-heading"><a class="rsswidget" href="https://groups.google.com/forum/feed/nature-protocols-discussion/topics/rss.xml?num=15"><img class="rss-widget-icon" style="border:0" width="14" height="14" src="https://blogs.nature.com/stepwise/wp-includes/images/rss.png" alt="RSS" /></a> <a class="rsswidget" href="">Recent Discussions</a></h2></div><div id="wpn-rss-5" class="wpn-widget wpn-widget-rss wpn-"><h2 class="box-heading"><a class="rsswidget" href="https://www.nature.com/protocolexchange/protocols.rss?protocol_search%5Bfacets%5D%5Bderived-protocol-type%5D=Community+Contributed"><img class="rss-widget-icon" style="border:0" width="14" height="14" src="https://blogs.nature.com/stepwise/wp-includes/images/rss.png" alt="RSS" /></a> <a class="rsswidget" href="">Latest from Protocol Exchange</a></h2></div><div id="wpn-hybrid-categories-3" class="wpn-widget wpn-categories wpn-"><h2 class="box-heading">Categories</h2><ul class="xoxo categories"><li class="cat-item cat-item-39"><a href="https://blogs.nature.com/stepwise/category/hints-and-wrinkles" >Hints and Wrinkles</a></li><li class="cat-item cat-item-40"><a href="https://blogs.nature.com/stepwise/category/news-and-updates" >News and Updates</a></li><li class="cat-item cat-item-41"><a href="https://blogs.nature.com/stepwise/category/spotlights" >Spotlights</a></li><li class="cat-item cat-item-42"><a href="https://blogs.nature.com/stepwise/category/there-and-back-again" >There and Back Again</a></li><li class="cat-item cat-item-1"><a href="https://blogs.nature.com/stepwise/category/uncategorized" >Uncategorized</a></li></ul></div><div id="wpn-npg-blogroll-widget-2" class="wpn-widget wpn-npg-blogroll-widget wpn-"><h2 class="box-heading">Blogs we like</h2><ul><li><p class="wpn-catline"><span>Blogs we like</span></p><h2><a href="https://blogs.nature.com/from_the_lab_bench/" title="A Nature Network blog by Paige Brown" target="_blank">From the Lab Bench</a></h2><p class="wpn-description">A Nature Network blog by Paige Brown</p></li> <li><p class="wpn-catline"><span>Blogs we like</span></p><h2><a href="https://blogs.nature.com/methagora" title="A blog from Nature Methods">Methagora</a></h2><p class="wpn-description">A blog from Nature Methods</p></li> <li><p class="wpn-catline"><span>Blogs we like</span></p><h2><a href="https://orgprepdaily.wordpress.com/">Org Prep Daily</a></h2></li> <li><p class="wpn-catline"><span>Blogs we like</span></p><h2><a href="https://blogs.nature.com/thescepticalchymist" title="A blog by Nature Chemistry ">The Sceptical Chymist</a></h2><p class="wpn-description">A blog by Nature Chemistry </p></li> <li><p class="wpn-catline"><span>Blogs we like</span></p><h2><a href="https://blogs.nature.com/tradesecrets/" title="A blog from Bioentrepreneur">Trade Secrets</a></h2><p class="wpn-description">A blog from Bioentrepreneur</p></li> </ul></div><div id="wpn-npg-natureblogs-links-widget-2" class="wpn-widget wpn-npg-natureblogs-links-widget wpn-">