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Assuming this is a symmetric membrane protein with at least 3-fold rotational symmetry, you don’t need the top and bottom views to fully sample Fourier space and arrive at a high-quality reconstruction. Side-views of a rotationally symmetric particle are sufficient,
and the reconstruction will be complete.
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Dmitry Lyumkis</div>
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The Salk Institute for Biological Studies</div>
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E: <a href="mailto:dlyumkis@salk.edu" class="">dlyumkis@salk.edu</a></div>
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<div class="">On Mar 24, 2020, at 10:53 PM, Jacopo Marino <<a href="mailto:jacopo.marino@psi.ch" class="">jacopo.marino@psi.ch</a>> wrote:</div>
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<p class=""><tt class="">How much do you really need top and bottom views for 3D reconstruction ? Have you tried ?</tt><br class="">
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<div class="moz-cite-prefix">On 25.03.2020 06:48, Saumya Verma Bajaj wrote:<br class="">
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<font style="" face="arial, sans-serif" class="">Dear all,</font></div>
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<font face="arial, sans-serif" class="">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.
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<font face="arial, sans-serif" class="">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.
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<font face="arial, sans-serif" class="">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></div>
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<font face="arial, sans-serif" class="">Any suggestions to salvage this set of data will be very helpful.</font></div>
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<font face="arial, sans-serif" class="">Thank you.</font></div>
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<font face="arial, sans-serif" class="">Best regards,</font></div>
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<font style="" face="arial, sans-serif" class="">Saumya</font></div>
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<pre class="moz-signature" cols="72">--
Dr. Jacopo Marino
Laboratory of Biomolecular Research
Paul Scherrer Institute
OFLB/005
5232 Villigen PSI, Switzerland
tel: +41 56 310 5484 (or +41 56 310 5777)
e-mail: <a class="moz-txt-link-abbreviated" href="mailto:jacopo.marino@psi.ch">jacopo.marino@psi.ch</a>
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