Hi Julian, 

This can also be perfumed within Frealign (and maybe the latest version of cisTEM?). We described the application of a focused classification approach on an individual asymmetric subunit basis here: 

Passos, D. O., Li, M., Yang, R., Rebensburg, S. V., Ghirlando, R., Jeon, Y., et al. (2017). Cryo-EM structures and atomic model of the HIV-1 strand transfer complex intasome. Science, 355(6320), 89–92. http://doi.org/10.1126/science.aah5163

And the evaluation of distinct focused classification strategies (with symmetry expansion) here: 

Zhang, C., Cantara, W., Jeon, Y., Musier-Forsyth, K., Grigorieff, N., & Lyumkis, D. (2018). Analysis of discrete local variability and structural covariance in macromolecular assemblies using Cryo-EM and focused classification. Ultramicroscopy. http://doi.org/10.1016/j.ultramic.2018.11.016

Best, 

Dmitry


Dmitry Lyumkis
Assistant Professor
The Salk Institute for Biological Studies
10010 North Torrey Pines Road, La Jolla, CA 92037
T: 858-453-4100 x1155






On Mar 5, 2019, at 2:39 AM, Charles Bayly-Jones <[log in to unmask]> wrote:

Yes, the relion_particle_symmetry_expand function will allow you to accomplish this. Then perform masked 3D classification as you described. It is very useful indeed :) 

You could also use the localised reconstruction method from  Ilca et al. (2015)  doi:10.1038/ncomms9843 

Best of luck,
Charlie
_________________

Charles Bayly-Jones
BSc(ScSchProg)(Hons)

PhD Candidate
Teaching Associate

Monash Biomedicine Discovery Institute
Department of Biochemistry and Molecular Biology
Level 2, Building 77
23 Innovation Walk
Clayton VIC 3800
Australia


On Tue, 5 Mar 2019 at 21:01, Julian Reitz <[log in to unmask]> wrote:
Dear all,

we are working on a dataset from a homo-trimeric protein. Until now we have very successfully refined the structure to good resolution applying C3 symmetry in RELION.

However, we also have a sample from this protein that contains a low affinity binding substrate and we would like to do a classification to see if there are some subunits showing the expected conformational change due to substrate binding. The point is, that we do not expect the ligand to bind to all three monomers of one particle but that the conformational change might only happen in one of the monomers of a particle. Therefore, normal 3D-classification seems unrewarding and a masked classification would suffer from the aspect, that we always only look on one of the monomers ignoring two-thirds of the data.

We were thinking that it might be helpful if we somehow triplicate the particles, each rotated by 120° to get all of the three monomers from one particle inside the mask. Is there any clever way to do this in RELION or does anybody else has a good idea how we could proceed?

Best,
Julian

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