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If it’s a twofold rotationally symmetric object, adhered to the air-water interface along a single “side" view, then a projection along, e.g., phi=0/theta=90 samples the same Fourier plane as the second projection along phi=180/theta=90. The reconstruction
will behave in a manner that is identical to one that is composed of exclusively “top” views of a rotationally symmetric object (or, more simply, an asymmetric particle with one preferential orientation, normalizing for the number of asymmetric units). Basically,
you’ll end up hyper-sampling around one plane in both cases, except that the planes will lie along distinct axes of the transform. Both are bad cases and, in the absence of other views, will lead to a bad reconstruction. To be clear: this is assuming that
there’s only one preferential orientation along the side view of a two-fold rotationally symmetric particle, and not more. If you have 3-fold rotational symmetry, and the sample is adhered to its “side” view, projections separated by phi=120° spacing will
lead to sampling of Fourier planes separated by phi=60° (due to symmetry). Effectively, you add two additional planes. Assuming you have enough particles, and there’s a bit of spread in the phi angle, in most practical cases, the reconstruction of a 3-fold
rotationally symmetric object will be complete (or nearly so). The higher the rotational symmetry, obviously the better.
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<div class="">To answer the original question, adding top views for your membrane protein will be negligible in the reconstruction. Looks like you should already get a very nice map from the current data. Give it a try. </div>
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<div class="">Best, </div>
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<div class="">Dmitry </div>
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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 25, 2020, at 12:06 AM, Philip Köck <<a href="mailto:koeck@kth.se" class="">koeck@kth.se</a>> wrote:</div>
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