Hi Ufuk,
You could do symmetry expansion in RELION, then pick one nanobody
binding site at one of your asymmetric units and create a mask for it,
then use focussed 3D classification without alignment - hopefully you
can then find classes with and without nanobody. You should then have
one asymmetric unit on your particle with close to 100 % nanobody
occupancy, and can then proceed with refinement as normal (but without
applying symmetry). You may have to increase the T-value in the 3D
classification a lot, as it sounds like one nanobody will be a small
portion of your box.
Cheers,
Alex
--
Dr Alexander Cook
Postdoctoral Research Associate | Higgins Lab *
BTP Junior Research Fellow in Biochemistry | Pembroke College +
* Department of Biochemistry, University of Oxford
+ University of Oxford
On 28/07/2022 10:43, Ufuk Borucu wrote:
> Hello,
>
> We have data on a particle that has a core with icosahedral symmetry. This core is decorated with a variable number of nanobodies that bind to icosahedral monomers at variable locations. We can't get a fully decorated core due to steric clashes (most likely). We are most interested in resolving the structure of a monomer (70 kDa) bound with the nanobody (17 kDa). We do not necessarily need to resolve the structure of the entire complex (core + nanobodies). Does anyone please have suggestions how we should best go about solving this problem?
>
> Many thanks in advance,
> Ufuk
>
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