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Hardware developments to improve image quality for cryoEM of biological specimens


Type

Thesis

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Authors

Dickerson, Joshua 

Abstract

Several advances in cryoEM hardware have been integral in transforming the technique into a standard technique for high-resolution macromolecular structure determination. We are now entering an era where we can experimentally determine or predict the structure of almost the entire proteome. Efforts are naturally moving from solving purified structures to visualising macromolecules inside cells. However, because these specimens are an order of magnitude thicker than purified specimens, cryoEM inside cells is restricted to large complexes, such as ribosomes. Here, I investigate the use of two hardware developments, liquid helium cooling of the specimen during imaging and chromatic aberration (Cc) correction, to improve the quality of cryoEM images, the latter in particular for thick specimens. I demonstrate that Cc correction can be used to correctly focus inelastically scattered electrons, enhancing the signal significantly for thick specimens. I also demonstrate that reducing the specimen temperature from 81 K to 13 K reduces the rate of radiation damage in single-particle cryoEM, and I present progress in understanding other changes that occur when irradiating specimens at these temperatures. Lastly, I calculate the impact that these hardware developments could have on the minimum molecular mass of protein that we can visualise in situ by cryoEM. Taken together with other hardware and software developments, these technologies could dramatically alter the landscape of in situ cryoEM, with proteins as small as 100 kDa potentially being within reach.

Description

Date

2023-12-17

Advisors

Russo, Christopher

Keywords

Cc correction, cryoEM, Liquid Helium, Radiation Damage, Structural Biology, TEM

Qualification

Doctor of Philosophy (PhD)

Awarding Institution

University of Cambridge