Affinity cryo-electron microscopy: Methods development and applications

Guimei Yu, Purdue University

Abstract

Single particle cryo-electron microscopy (cryo-EM) is an emerging powerful tool for structural studies of macromolecular assemblies. Although less concentrated and smaller amounts of samples are required for single particle cryo-EM compared to X-ray crystallography, it remains challenging to study specimens that are low-abundance, low-yield, or short-lived. The recent development of affinity grid techniques holds great promise to tackle these challenging samples by combining the sample purification and freezing on TEM grids steps in cryo-EM grid preparation into a single step, revolutionize the grid preparation of cryo-EM, and extend single particle cryo-EM to a routine structural biology tool to characterize structures of a broad spectrum of macromolecules. ^ In my PhD study, I have established a new design of the affinity cryo-EM approach, cryo-SPIEM that applies a traditional pathogen diagnosis tool Solid Phase Immune Electron Microscopy (SPIEM) to the single particle cryo-EM method, and also systematically explored the applications of affinity EM approaches and the potentials of affinity cryo-EM approaches for near-atomic single particle 3-D reconstruction. The cryo-SPIEM approach provides an alternative, largely simplified and easier to use affinity grid that directly works with most native macromolecular complexes with established antibodies, and enables cryo-EM studies of native samples directly from cell cultures. The application of the affinity cryo-EM approach for high-resolution cryo-EM has been demonstrated successfully by solving a 6.3 Å structure of Tulane virus using the polylysine-based affinity grid, and a 2.6 Å structure of Tulane virus using the antibody-based affinity grid with a sample of low concentrations that defies standard cryo-EM study. Moreover, we have applied the affinity grid technique to investigate the interaction between Tulane virus and its cellular receptor Histo-blood group antigens, which revealed the potential roles of HBGA receptors in mediating genome release.^

Degree

Ph.D.

Advisors

Wen Jiang, Purdue University.

Subject Area

Molecular biology|Microbiology|Biophysics

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