Technology Development for 3D Electron Microscopy

Division Of Basic Sciences - Nci

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Abstract

Cryo-EM technology has now become firmly rooted as an indispensable tool in structural biology. Starting from 2015, when we reported the first high resolution structure of a protein by single particle cryo-EM to our recent work in 2018 further advancing resolutions to much better than 2 Angstroms, we have been both developing and applying cryo-EM technology to study protein conformations and to map drug binding sites on targets of medical relevance. These advances have required many methodological adjustments in specimen preparation, data collection and image processing and al of these advances have been essential for the progress we have made on several biologically interesting projects over the last year. Two examples not covered in other sections describing work in the lab are highlighted below. Prokaryotic cells possess CRISPR-mediated adaptive immune systems that protect them from foreign genetic elements, such as invading viruses. A central element of this immune system is an RNA-guided surveillance complex capable of targeting non-self DNA or RNA for degradation in a sequence- and site-specific manner analogous to RNA interference. Although the complexes display considerable diversity in their composition and architecture, many basic mechanisms underlying target recognition and cleavage are highly conserved. In a paper published in the journal Cell in December 2017, we used cryo-EM to show that the binding of target double-stranded DNA (dsDNA) to a Type I-F Csy surveillance complex leads to large quaternary and tertiary structural changes in the complex that are likely necessary in the pathway leading to target dsDNA degradation by a trans-acting helicase-nuclease. Comparison of the structure of the surveillance complex before and after dsDNA binding, or in complex with three virally-encoded anti-CRISPR suppressors that inhibit dsDNA binding, revealed mechanistic details underlying target recognition and inhibition. Accurate chromosome segregation requires the proper assembly of kinetochore proteins. A key step in this process is the recognition of the histone H3 variant CENP-A in the centromeric nucleosome by the kinetochore protein CENP-N. In a paper published in the journal Science in January 2018, we reported cryo-EM, biophysical, biochemical, and cell biological studies of the interaction between the CENP-A nucleosome and CENP-N. We showed that human CENP-N confers binding specificity through interactions with the L1 loop of CENP-A, stabilized by electrostatic interactions with the nucleosomal DNA. Mutational analyses demonstrated analogous interactions in Xenopus, which is further supported by residue-swapping experiments involving the L1 loop of CENP-A. Our results are consistent with co-evolution of CENP-N and CENP-A, and establish the structural basis for recognition of the CENP-A nucleosome to enable kinetochore assembly and centromeric chromatin organization.