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<p class="MsoNormal" style="line-height:16.0pt;mso-line-height-rule:exactly"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Dear All,<br>
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<p class="MsoNormal" style="line-height:16.0pt;mso-line-height-rule:exactly"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">The currently prevalent single-particle resolution assessment methodology is based on Fourier Shell Correlation (FSC).<span style="mso-spacerun:yes">
</span>However, its use remains controversial as the outcome is subjective, mainly due to the necessity to use smooth-edged masks, which introduce artifacts and make it difficult to support the outcome by statistical analysis.</span></p>
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<p class="MsoNormal" style="line-height:16.0pt;mso-line-height-rule:exactly"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">In the article Reliable cryo-EM Resolution Estimation with Modified FSC (mFSC) that just appeared on IUCrJ site (<a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__doi.org_10.1107_S2052252520011574&d=DwMDaQ&c=bKRySV-ouEg_AT-w2QWsTdd9X__KYh9Eq2fdmQDVZgw&r=yEYHb4SF2vvMq3W-iluu41LlHcFadz4Ekzr3_bT4-qI&m=8P5zavyLYwIWiwPar72EogSpE8PJtfj-B0muENTEnAI&s=Ntn8H-2368avF171GWdLgm2KBTbOhp7T_0jw6Yj8P-U&e=">https://doi.org/10.1107/S2052252520011574</a>)
I addressed these deficiencies and modified the way FSC is computed.<span style="mso-spacerun:yes">
</span>As a result, the proposed mFSC is free from mask-induced artifacts and thus only binary masks outlining region of interest are used.<span style="mso-spacerun:yes">
</span>Moreover, this modification makes it possible to compute the number of degrees of freedom associated with mFSC coefficients and thus assess the resolution using one-sided Confidence Interval (CI). The use of statistical test prevents users from accepting
unreliable results that may stem from using excessively small regions of interest, as in local resolution analysis.<span style="mso-spacerun:yes">
</span>The mFSC program is available at <a href="https://urldefense.com/v3/__http://sparx-em.org/sparxwiki/sxresolution__;!!Mih3wA!U-Ahz68KwuVPUNP6BMZ1CSsJVoTLABdO9IdUnCsBWHrnzowqgjv-nocb8RZ6sdpyyg$">http://sparx-em.org/sparxwiki/sxresolution</a>.</span></p>
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<p class="MsoNormal" style="line-height:16.0pt;mso-line-height-rule:exactly"><span style="font-size:11.0pt;font-family:"Arial",sans-serif">The mFSC is particularly effective in addressing a number of problems:</span></p>
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<li><span style="font-size:11.0pt;font-family:"Arial",sans-serif">Since there are no mask-induced artifacts, it is possible to assess resolution by entirely eliminating solvent regions, i.e., to use masks that coincide with the structure itself<br>
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</span></li><li><span style="font-size:11.0pt;font-family:"Arial",sans-serif">I show that local resolution analysis is of limited value as the spatial resolvability of the outcome is poor. Instead, I suggest using mFSC to compute resolution of biologically relevant segments
of the structure or even its secondary elements, such as helices.<br>
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</span></li><li><span style="font-size:11.0pt;font-family:"Arial",sans-serif">mFSC makes it possible to compute artifact-free resolution curves for helical structures (for which standard FSC in most cases fails due to reciprocal space artifacts)<br>
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</span></li><li><span style="font-size:11.0pt;font-family:"Arial",sans-serif">The Confidence Interval-based mFSC all but eliminates subjectiveness of resolution assessment.</span></li></ul>
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<p>Regards,<br>
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<div class="PlainText"> <br>
Pawel A. Penczek, Ph.D.<br>
Professor<br>
Department of Biochemistry and Molecular Biology<br>
The University of Texas - McGovern Medical School<br>
MSB 6.220<br>
6431 Fannin<br>
Houston, TX 77030<br>
USA<br>
<br>
phone: 713-500-5416<br>
fax: 713-500-6297<br>
pawel.a.penczek@uth.tmc.edu<br>
<a href="https://urldefense.com/v3/__http://www.uth.tmc.edu/bmb/faculty/pawel-penczek.html__;!!Mih3wA!U-Ahz68KwuVPUNP6BMZ1CSsJVoTLABdO9IdUnCsBWHrnzowqgjv-nocb8RZcF7IYlA$">http://www.uth.tmc.edu/bmb/faculty/pawel-penczek.html</a></div>
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