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<p>Hi Saumya,</p>
<p>As others have remarked too: it actually doesn't look so bad and
you don't really need the top views. I would try and combine
particles from basically all the 2D classes that you show
(possibly excluding some from the bottom 2 rows, but that will not
matter much) for a 3D initial model generation (if you don't have
a model yet) and then refine or 3d-classify with the expected
symmetry (C4?). You might be surprised.</p>
<p>HTH,</p>
<p>Sjors</p>
<p><br>
</p>
<div class="moz-cite-prefix">On 25/03/2020 05:48, Saumya Verma Bajaj
wrote:<br>
</div>
<blockquote type="cite"
cite="mid:CA+_vNvRA9sTiqTN4Pk2wRuROCPJJap9nGQi-_ToFSqAhCTSFpQ@mail.gmail.com">
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<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font style=""
face="arial, sans-serif">Dear all,</font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif"> </font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif">I am trying to solve the structure
of a tetrameric membrane protein
complex, with the protein embedded in detergent micelle
(0.05% GDN) and a
soluble accessory protein attached to it. </font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif"> </font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif">Following 2D classification, while
the side views and oblique views are
easily visible, and the top/bottom are very few (~1-2%)
(please see attached image)
and get further diminished in subsequent rounds of 2D
classification. </font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif"> </font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif">While we are trying different grid
types to overcome the orientation
problem at the sample level, I was wondering if there are
certain tweaks we can
make to the analysis parameters (particle picking, box size,
mask, ctf etc.) to
enhance the signal of protein embedded within a micelle in
the current data
set. We are using Relion3.1 for SPA.</font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif"> </font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif">Any suggestions to salvage this set
of data will be very helpful.</font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif"> </font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif">Thank you.</font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font
face="arial, sans-serif">Best regards,</font></p>
<p class="MsoNormal" style="margin:0cm 0cm
0.0001pt;line-height:normal;text-align:justify"><font style=""
face="arial, sans-serif">Saumya</font></p>
</div>
<br>
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