2024Suder Workflow

Citation

Suder, Diana S. / Gonen, Shane. Mitigating the Blurring Effect of CryoEM Averaging on a Flexible and Highly Symmetric Protein Complex through Sub-Particle Reconstruction. 2024. Intl. J. Molecular Sciences, Vol. 25, No. 11, p. 5665

Abstract

Many macromolecules are inherently flexible as a feature of their structure and function. During single-particle CryoEM processing, flexible protein regions can be detrimental to highresolution reconstruction as signals from thousands of particles are averaged together. This “blurring” effect can be difficult to overcome and is possibly more pronounced when averaging highly symmetric complexes. Approaches to mitigating flexibility during CryoEM processing are becoming increasingly critical as the technique advances and is applied to more dynamic proteins and complexes. Here, we detail the use of sub-particle averaging and signal subtraction techniques to precisely target and resolve flexible DARPin protein attachments on a designed tetrahedrally symmetric protein scaffold called DARP14. Particles are first aligned as full complexes, and then the symmetry is reduced by alignment and focused refinement of the constituent subunits. The final reconstructions we obtained were vastly improved over the fully symmetric reconstructions, with observable secondary structure and side-chain placement. Additionally, we were also able to reconstruct the core region of the scaffold to 2.7 Å. The data processing protocol outlined here is applicable to other dynamic and symmetric protein complexes, and our improved maps could allow for new structure-guided variant designs of DARP14.

Links

https://www.mdpi.com/1422-0067/25/11/5665