<html>
<head>
<meta http-equiv="Content-Type" content="text/html; charset=utf-8">
<style type="text/css" style="display:none;"><!-- P {margin-top:0;margin-bottom:0;} --></style>
</head>
<body dir="ltr">
<div id="divtagdefaultwrapper" style="font-size:12pt;color:#000000;font-family:Calibri,Helvetica,sans-serif;" dir="ltr">
<p style="margin-top:0;margin-bottom:0">Dear Mohamed,</p>
<p style="margin-top:0;margin-bottom:0"><br>
</p>
<p style="margin-top:0;margin-bottom:0">If you see fewer particles in ice after adding detergent to the sample, it can help to apply and blot your sample on the grid multiple times before you plunge-freeze. We described the procedure some time ago here:</p>
<p style="margin-top:0;margin-bottom:0"><a href="https://www.sciencedirect.com/science/article/pii/S104784771730031X" class="OWAAutoLink" id="LPlnk17595" previewremoved="true">https://www.sciencedirect.com/science/article/pii/S104784771730031X</a></p>
<p style="margin-top:0;margin-bottom:0">I had the exact issue you described with an Epstein Barr Virus protein complex. Adding the detergent (0.01% NP40 in my case) improved the orientations for me, and multiple rounds of blotting improved the numbers of particles
in ice. We have also seen it work with DDM detergent. Hope this trick also works for you, good luck!</p>
<p style="margin-top:0;margin-bottom:0"><br>
</p>
<p style="margin-top:0;margin-bottom:0">Best,</p>
<p style="margin-top:0;margin-bottom:0"><br>
</p>
<p style="margin-top:0;margin-bottom:0">Joost</p>
</div>
<hr style="display:inline-block;width:98%" tabindex="-1">
<div id="divRplyFwdMsg" dir="ltr"><font face="Calibri, sans-serif" style="font-size:11pt" color="#000000"><b>From:</b> 3dem <3dem-bounces@ncmir.ucsd.edu> on behalf of Mohamed A Sayed <mohamed.sobhy@kaust.edu.sa><br>
<b>Sent:</b> Thursday, May 30, 2019 7:26:01 PM<br>
<b>To:</b> 3dem@ncmir.ucsd.edu<br>
<b>Subject:</b> [3dem] question about preferred orientation</font>
<div> </div>
</div>
<style type="text/css" style="display:none">
<!--
p
{margin-top:0px;
margin-bottom:0px}
-->
</style>
<div dir="ltr" style="font-size:12pt; color:#000000; background-color:#FFFFFF; font-family:Calibri,Arial,Helvetica,sans-serif">
<p>If you would please advise on the following:<br>
</p>
<p>I have protein that shows strong preferential orientation. The buffer only contains (20 mM Tris. HCl pH7.5, 150 mM NaCl). </p>
<p>I tried adding detergent CHAPS (8 mM final concentration) and the number of particles dropped to only a few per hole and most of the protein particels were on the carbon film. <span style="font-family:Calibri,Arial,Helvetica,sans-serif; font-size:16px; background-color:rgb(255,255,255)">I
tried different hole size gold grids r</span><span style="font-family:Calibri,Arial,Helvetica,sans-serif; font-size:16px; background-color:rgb(255,255,255)">1.2/1.3</span><span style="font-family:Calibri,Arial,Helvetica,sans-serif; font-size:16px; background-color:rgb(255,255,255)"> and
r</span><span style="font-family:Calibri,Arial,Helvetica,sans-serif; font-size:16px; background-color:rgb(255,255,255)">2/2 but the same result. Any advice how to obtain random distribution in such case.</span></p>
<p><br>
</p>
<p>Is there an adjustment in Relion 3d classification parameters in order to obtain a 3d classification? I tried without symmetry and both the initial model and 3d classifcation fail. <br>
</p>
<p><span style="font-size:12pt">number of classes</span><br>
</p>
<p><span style="font-size:11.5pt; line-height:107%; font-family:"Segoe UI",sans-serif; color:#212121">Initial angular sampling</span><br>
</p>
<p>symmetry<br>
</p>
<p>CTF correction or 'Ignore CTF’s until first peak'.</p>
<p><span style="font-size:12pt">'Initial angular sampling'.</span></p>
<p>mask<br>
</p>
<p>SGD:<br>
</p>
<p>Final resolution<br>
</p>
<p>Initial mini-batch size<br>
</p>
<p><span style="font-size:11.5pt; line-height:107%; font-family:"Segoe UI",sans-serif; color:#212121"><br>
</span></p>
<p><span style="font-family:Calibri,Arial,Helvetica,sans-serif; font-size:16px; background-color:rgb(255,255,255)">Thank you very much for your help.</span><br>
</p>
</div>
</body>
</html>