Improving electron microscopic hardware to push the resolution limits in single particle cryo-EM

by Ka Man Yip
Doctoral thesis
Date of Examination:2022-05-05
Date of issue:2022-07-19
Advisor:Prof. Dr. Holger Stark
Referee:Prof. Dr. Kai Tittmann
Referee:Dr. Michael Habeck
Referee:Prof. Dr. Jörg Enderlein
Referee:Prof. Dr. Bert De Groot
Referee:Dr. Alexis Caspar Faesen
Persistent Address: http://dx.doi.org/10.53846/goediss-9348

 

 

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Abstract

English

Electron cryomicroscopy (cryo-EM) is a powerful tool in structural biology for determining 3D structures of macromolecular complexes. In the previous decade, many technological advancements have been introduced to improve the methodology of single particle cryo-EM, and hence the resolution of its protein reconstructions. These advancements include developments in both software algorithms and microscope hardware. Despite all these developments, the resolution of single particle cryo-EM cannot surpass 1.5 Å, a barrier blocking direct visualization of atomic details of the protein structure. Here, we show that by improving the optical performance of the electron microscope with new hardware, such resolution barrier can be removed, and atomic resolution of 1.25 Å can be reached with an engineered protein apoferritin. The atomic resolution protein structure helped set benchmarks for validating data quality in high resolution cryo-EM. We found that the new microscope also benefited to other proteins with lower contrast and higher flexibility, such as proteasome, chaperonin and fatty acid synthetase. For these proteins, sample properties and behavior were the major limitations. We anticipate that this would be alleviated by introducing other new microscope hardware, so that in the future, time and effort of structural biologists can concentrate on biochemical preparation of the sample, and single particle cryo-EM can routinely yield high quality protein structures at atomic resolution.
Keywords: cryo-EM; single particle analysis; transmission electron microscopy; monochromator; aberration corrector; energy filter
 
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