Direct Visualization of a 26 kDa Protein by Cryo-Electron Microscopy Aided by a Small Scaffold Protein

Abstract

Cryo-electron microscopy (cryo-EM)-based structure determination of small proteins is hindered by the technical challenges associated with low signal-to-noise ratios of their particle images in intrinsically noisy micrographs. One solution is to attach the target protein to a large protein scaffold to increase its apparent size and, therefore, image contrast. Here we report a novel scaffold design based on a trimeric helical protein, E. coli ornithine transcarbamylase (OTC), fused to human ubiquitin. As a proof of principle, we demonstrated the ability to resolve a cryo-EM map of a 26 kDa human ubiquitin C-terminal hydrolase (UCHL1) attached to the C-terminus of ubiquitin as part of the trimeric assembly. The results revealed conformational changes in UCHL1 upon binding to ubiquitin, namely, a significant displacement of α-helix 2, which was also observed by X-ray crystallography. Our findings demonstrated the potential of the trimeric OTC scaffold design for studying a large number of ubiquitin interacting proteins by cryo-EM.

Genetically Encoded Crosslinking Enables Identification of Multivalent Ubiquitin‐Deubiquitylating Enzyme Interactions

“…Its interaction is structurally well characterized with the Ub-bound structure having been solved by both X-ray crystallography and cryo-electron microscopy (CryoEM). [28,40] Our Ub T9Bpa crosslinking results show that there is one equivalent of Ub binding as suggested by SDS-PAGE gel. Photocrosslinking reaction mixtures were subjected to tryptic digestion and analysis by LC-MS/MS.…”

Heterologous expression of naturally evolved putative de novo proteins with chaperones

“…Soluble expression and purification will be crucial for structural characterization via CD, NMR, and Cryo-EM. Due to their small size and high disorder content, only NMR 25 and potentially Cryo-EM 64 will be capable of solving the structure of de novo proteins experimentally. Even in light of the recent dawn of computational structure prediction, 65,66 experimental structural and functional determination remains necessary, especially for de novo proteins.…”

The CryoEM structure of human serum albumin in complex with ligands

“…Conclusions. While cryoEM structures of molecules smaller that 100 kDa in size are available [25][26][27][28]31 they have been often the results of a one-off process that tested the current technology and software, or obtained by extensive manipulation of the sample itself 26,[47][48][49] but did not address the need for reproducibility in structure determination, as well as the necessity of identifying ligands bound, two essential requirement for the use of cryoEM derived information in drug discovery and development. In this paper we have shown that routine structural determination of molecules less than 70 kDa in size is possible, and that visualization of bound ligands is achievable.…”