Hi,
You might find this comment from Sjors useful.
https://www.jiscmail.ac.uk/cgi-bin/webadmin?A2=ind1908&L=CCPEM&O=D&P=114682
Best regards,
Takanori Nakane
> Hi,
> I have a problem with aligning particles of my filament structure using
Reli
> on 3.0.5. This filamentous protein is expected to be mostly b-strands
and ha
> s an estimated helical twist close to 180°. Because of the diameter of the
> filament and few repeats in the box, the helical reconstruction is
problemat
> ic. Thus, I have decided to use a single particle approach with
particles wh
> ich were picked as helical fragments. After 2D classification, I have
very g
> ood 2D classes with a lot of features, but the problem starts in 3D
classifi
> cation.
>
> ~400k particles 2x binned of 106 px (2.16 A/px) were classified in 3D to
5 c
> lasses (3.7 angular sampling), where 2 classes gave ~8 A resolution
models w
> ith expected features. Each class was refined against the 3D class
model, bu
> t Refine3D gave nonsense results in both cases and the previously seen
featu
> res were gone. I also tried the 3D refinement without angular sampling
and o
> nly local searches (0.9°) but results were similar. I tried different maski
> ng, repeating 3D classification from previous classes, but no
improvement wa
> s observed.
>
> As a proof of principle, extracted particles from Relion were imported as
> particle stacks to Cryosparc v2. After ab initio, 2D, and 3D
> classifications, the refinement of particles gave very good map quality at
> the ~Nquist frequency (~4.5 A). Because Cryosparc, in general,
> overestimates the resolution, the local resolution was calculated and was
> in the range of 5-12 A.
>
> The question is how to force Relion to align particles as Cryosparc did? I
> also exported particles used by Cryosparc in the final refinement and
> reimported them back to Relion, but the alignment problem was still
> observed. I tried different T values in 3D classification (1, 4, 25) but
> with the same outcome.
> Would you suggest any other tricks which would help with the alignment of
> particles in Relion?
> I would really appreciate any insights you have.
>
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