[3dem] 3D classification

[Ali Punjani]

Hi Eric,

As a direct method to resolve the heterogeneity, you may wish to try the
new 3D Variability Analysis method available in cryoSPARC v2.9 (released
earlier this month). This algorithm can detect very small conformational
changes at high resolution, by computing the eigenvectors of the 3D density
covariance of the density. These eigenvectors correspond to the modes of
maximal variability in the particle data, and therefore can be used to
resolve both discrete and continuous heterogeneity. More details, including
examples and a detailed tutorial can be found here:
https://cryosparc.com/docs/tutorials/3d-variability-analysis/
3D Variability Analysis can also be used with a mask, to focus on the
relevant region of the structure, ignoring conformational variability that
may be occurring in other irrelevant regions (e.g. it can help to mask out
the micelle or nanodisc for a membrane protein).

Note that, as Sjors already mentioned, the major determinant of success
will be detectability of the change caused by binding. If there is no
conformational change associated with the binding, it is unlikely that the
ligand alone would cause enough change in each particle image to be
statistically distinguishable from noise, regardless of the computational
technique used.

Good luck with your processing!
Ali


It can also be used in a masked fashion,
On Thu, Jul 11, 2019 at 8:24 PM Eric Hanssen <ehanssen at unimelb.edu.au>
wrote:

> Hi all,
>
> Before I commit some computer resource to a job I’d rather know if it is
> doable.
>
>
>
> I have 200,000 particle of a 600kDa protein, a current map at 3.4A that
> looks very nice, I do not need a better resolution for now. I have a drug
> (dipeptide) attached to the protein but  only a subset of the protein has
> the drug bound. Is it possible with a 3D classification to separate such a
> small difference? I don’t mind which software, relion, cryosparc, eman2 ….
>
>
>
> Or is there a better way, eg. 3D classification of a very small box size
> centered around the binding site?
>
>
>
> Cheers
>
> Eric
>
>
>
>
>
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>
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