Journal Article

2.8 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome using cryo-electron microscopy

Campbell, Melody G, Veesler, David, Potter, Clinton S, Cheng, Anchi, Carragher, Bridget

2015

Overview

Recent developments in detector hardware and image-processing software have revolutionized single particle cryo-electron microscopy (cryoEM) and led to a wave of near-atomic resolution (typically ∼3.3 Å) reconstructions. Reaching resolutions higher than 3 Å is a prerequisite for structure-based drug design and for cryoEM to become widely interesting to pharmaceutical industries. We report here the structure of the 700 kDa Thermoplasma acidophilum 20S proteasome (T20S), determined at 2.8 Å resolution by single-particle cryoEM. The quality of the reconstruction enables identifying the rotameric conformation adopted by some amino-acid side chains (rotamers) and resolving ordered water molecules, in agreement with the expectations for crystal structures at similar resolutions. The results described in this manuscript demonstrate that single particle cryoEM is capable of competing with X-ray crystallography for determination of protein structures of suitable quality for rational drug design. Proteins perform many critical tasks within cells, and to do so, they must first fold into specific shapes. Being able to visualize these shapes can help scientists to understand how proteins work, and help them create drugs that can interact with the proteins to treat diseases. The past few years have seen the rapid development of an imaging technique called single-particle cryo-electron microscopy (or cryoEM for short), and this technique is now increasingly used to investigate protein structures. First, proteins are embedded in a thin film of non-crystalline ice by rapidly cooling to around the temperature of liquid nitrogen (below −180°C). This traps the protein in the shape it has in solution. High-energy electrons are then transmitted through the protein sample and their interaction with the atoms in the protein is recorded by a direct electron camera. The analysis of a large series of images recorded in this way can be used to determine the approximate positions of the atoms in the protein. Previously, single-particle cryoEM techniques have not produced a detailed enough protein structure to be useful to scientists interested in drug development. By refining these techniques, Campbell, Veesler et al. have now obtained the most detailed cryoEM protein structure to date—a structure of an enzyme complex that helps get rid of proteins that are misfolded or that have become too abundant. The structure is so detailed that it reveals the shapes of some small groups of atoms that stick out from the sides of amino acids in the enzyme complex. (Amino acids are the building blocks of enzymes and all other proteins.) Moreover, the structure shows where individual water molecules are positioned around the protein. The level of detail in the structure produced by Campbell, Veesler et al. is high enough to be useful to drug researchers. Furthermore, because only 10% of the images Campbell, Veesler et al. collected were used to produce the structure, future work will investigate whether incorporating more of the images could reveal structures in even greater detail.

• Publisher: eLife Sciences Publications Ltd,eLife Sciences Publications, Ltd
• Subject: Archaeal Proteins - chemistry / Archaeal Proteins - ultrastructure / Automation / Biology / Biophysics and Structural Biology / Cryoelectron Microscopy - methods / Crystallography / Crystallography, X-Ray / Data collection / Data processing / direct detector / Drug development / Electron microscopy / Gold / Image processing / Imaging, Three-Dimensional / Microscopy / Models, Molecular / Pharmaceutical industry / Proteasome Endopeptidase Complex - chemistry / Proteasome Endopeptidase Complex - ultrastructure / Proteasomes / Protein Conformation / Proteins / Reproducibility of Results / Sensors / Short Report / Software / Standard deviation / T. acidophilum / Thermoplasma - metabolism / X-ray crystallography