Electron Diffraction for High-Resolution Structure Determination of Biomolecules

Authors:

Niko W. Vlahakis* (nwvlahakis@g.ucla.edu), Ambarneil Saha, Logan S. Richards, Maria D. Flores, Jose A. Rodriguez, and Todd O. Yeates

Institutions:

University of California–Los Angeles DOE Institute for Genomics and Proteomics

Goals

The team aims to develop electron diffraction (ED) methods to extract high-resolution structural information from biomolecules and to determine novel biomolecular structures, expanding phasing methods, and identifying the extent to which ED data can inform on emergent properties of structures. Current goals include resolving the presence of substrates bound to enzymes within nanocrystals, identifying significant differences in electron scattering originating from crystals of opposite chirality, deconvoluting nanoscale lattice imperfections to resolve structures from more homogenous subregions of a single crystal, and understanding the rates and impact of electron beam damage on biomolecular crystals during data collection. Ultimately, the project’s objectives are to address the remaining gaps where electron crystallography still lags behind its X-ray counterpart and develop methods and strategies for closing them such that accurate structural information can be learned, even from nanocrystals.

Abstract

Electron diffraction has recently emerged as a powerful means of elucidating atomic structures from 3D crystals on the scale of nanometers in size, circumventing the requirement of large, pristine crystals required for successful structure determination by X-ray diffraction. The group aims to develop ED methods further, such that high-resolution structural information about biologically compelling molecules may be extracted from a wider range of targets with greater confidence. The team will discuss work done to expand the scope of molecular targets for electron diffraction as well as the range of ways to retrieve phase information from ED data, through the determination of structures encompassing short oligopeptides, cyclic peptides, natural products, and proteins. The team also interrogates the extent to which ED can be expected to provide accurate information on certain granular but nonetheless critical qualities of a molecule’s structure, such as the presence and configuration of a substrate bound in an enzyme’s active site or a chiral molecule’s absolute configuration. To carry out these investigations, researchers employ data collection from two methods in parallel:

• continuous-rotation selected area diffraction (microED), which enables researchers to routinely determine structures and assess the quality of structural information readily accessible to the vast majority of current investigators in the field, and
• scanning nanobeam diffraction (4D-STEM), which allows researchers to correlate high resolution-information in real space and reciprocal space to visualize lattice heterogeneity within single crystals and potentially resolve structures from crystal sub-domains.

As biomolecules are almost ubiquitously sensitive to damage by the electron beam, researchers probe mechanisms of radiation damage experienced by nanocrystals during data collection with both methods, offering insights into how the final structures solved by ED are impacted by beam damage and enabling the development of strategies for minimizing the damage delivered during ED experiments. Lastly, the team will perform ongoing work to validate and innovate sample preparation and data acquisition strategies for resolving improved macromolecular structures by electron diffraction.

Funding Information

Research supported by the BER program of the DOE Office of Science, award DE-FC02-02ER63421.