[3dem] Alignment problem in phage processing

[Leonardo Talachia Rosa]

Dear 3DEM community,
I hope you are all well.

A couple of months ago we started a collaboration on the structure of
bacteriophages, a CryoEM approach very different from what I have done
before.
I am facing one very specific problem, in which I was hoping to benefit
from your experience, if you would be kind to share a bit of it.

We are trying to solve the structure of a podoviridiae isolated form
wastewater, and when I do the 2D classification and 3D refinement on the
capside (using eihter Cryosparc or relion), the much stronger signal of the
major components results in neglected signals for the portal and tail
regions. As a consequence, particles are aligned in the central symmetry of
the capsid, and the 3D density ends up with portal signals averaged out
between the two extremities, artificially giving a two-tailed phage (figure
in annex). This is surely artificial, as the raw micrographs very clearly
show a single tail per phage.

I already tried several approaches to solve this issue:
-Using a larger box size to account for more tail signal
-Doing a refinement in C1, followed by a 3D classification, either with or
without particle alignment
-Doing a local refinement on the capsid only
-artificially deleting one of the tails in the map and using this as an
input for another round of refinement
-playing with the T parameter in relion, rising it up to 15
-doing signal subtraction on the capsid signal

I´m really out of ideas on how to solve this, so I thank you in advance for
your time, and for your willingness to share your views on it,
Kind wishes,
Leonardo

-- 
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Leonardo TALACHIA ROSA, PhD
PostDoctoral Researcher
Microbiology and Structural Biology
Instituto de Química, Universidade de São Paulo - IQ-USP
Av. Prof. Lineu Prestes, 748 - Butantã, São Paulo - SP
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