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Hi
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<div>there has to be some kind of misunderstanding here as in general it is impossible to perform eigeanalysis in the way described in the manual. <br>
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<div dir="ltr">Regards,
<div>Pawel Pawel</div>
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<div dir="ltr"><br>
On Jul 12, 2019, at 11:53 AM, Ali Punjani <<a href="mailto:alipunjani@cs.toronto.edu">alipunjani@cs.toronto.edu</a>> wrote:<br>
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<p><font color="Red" size="3" face="arial, sans-serif"><strong>**** EXTERNAL EMAIL ****</strong></font></p>
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<div dir="ltr">Hi Eric,<br>
<br>
As a direct method to resolve the heterogeneity, you may wish to try the new 3D Variability Analysis method available in cryoSPARC v2.9 (released earlier this month). This algorithm can detect very small conformational changes at high resolution, by computing
the eigenvectors of the 3D density covariance of the density. These eigenvectors correspond to the modes of maximal variability in the particle data, and therefore can be used to resolve both discrete and continuous heterogeneity. More details, including examples
and a detailed tutorial can be found here:
<div><a href="https://urldefense.proofpoint.com/v2/url?u=https-3A__cryosparc.com_docs_tutorials_3d-2Dvariability-2Danalysis_&d=DwMFaQ&c=bKRySV-ouEg_AT-w2QWsTdd9X__KYh9Eq2fdmQDVZgw&r=yEYHb4SF2vvMq3W-iluu41LlHcFadz4Ekzr3_bT4-qI&m=uikbWAmVC_qT6eK3I3JsUspENOZfAhHTGFHkCL4iUGQ&s=ij-rYNgTenO1SXXpZdrGsUv_6--ECLDYbb-Z2PRaX70&e=">https://cryosparc.com/docs/tutorials/3d-variability-analysis/</a></div>
<div>3D Variability Analysis can also be used with a mask, to focus on the relevant region of the structure, ignoring conformational variability that may be occurring in other irrelevant regions (e.g. it can help to mask out the micelle or nanodisc for a membrane
protein).<br>
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Note that, as Sjors already mentioned, the major determinant of success will be detectability of the change caused by binding. If there is no conformational change associated with the binding, it is unlikely that the ligand alone would cause enough change in
each particle image to be statistically distinguishable from noise, regardless of the computational technique used.</div>
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<div>Good luck with your processing!</div>
<div>Ali<br>
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It can also be used in a masked fashion,
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<div dir="ltr" class="gmail_attr">On Thu, Jul 11, 2019 at 8:24 PM Eric Hanssen <<a href="mailto:ehanssen@unimelb.edu.au">ehanssen@unimelb.edu.au</a>> wrote:<br>
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<p class="MsoNormal">Hi all, <u></u><u></u></p>
<p class="MsoNormal">Before I commit some computer resource to a job I’d rather know if it is doable.<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">I have 200,000 particle of a 600kDa protein, a current map at 3.4A that looks very nice, I do not need a better resolution for now. I have a drug (dipeptide) attached to the protein but only a subset of the protein has the drug bound.
Is it possible with a 3D classification to separate such a small difference? I don’t mind which software, relion, cryosparc, eman2 ….<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">Or is there a better way, eg. 3D classification of a very small box size centered around the binding site?
<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">Cheers<u></u><u></u></p>
<p class="MsoNormal">Eric<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><b><span style="font-size:10pt;font-family:"Comic Sans MS";color:navy">-----------------<u></u><u></u></span></b></p>
<p class="MsoNormal"><b><span style="font-size:10pt;font-family:"Comic Sans MS";color:navy">Assoc. Prof Eric Hanssen<u></u><u></u></span></b></p>
<p class="MsoNormal"><b><span style="font-size:10pt;font-family:"Comic Sans MS";color:navy"><u></u> <u></u></span></b></p>
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<p class="MsoNormal"><b><span style="font-size:10pt;font-family:"Comic Sans MS";color:rgb(31,73,125)">Honorary Principal Fellow – Department of Biochemistry and Molecular Biology<u></u><u></u></span></b></p>
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<p class="MsoNormal"><b><span style="font-size:10pt;font-family:"Comic Sans MS";color:rgb(31,73,125)">President Australian Microscopy and Microanalysis Society<u></u><u></u></span></b></p>
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