Single-particle cryo-EM at atomic resolution

Abstract

The three-dimensional positions of atoms in protein molecules define their structure and provide mechanistic insights into the roles they perform in complex biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more knowledge about protein function may be inferred. With breakthroughs in electron detection and image processing technology, electron cryomicroscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years 1,2 . However, obtaining cryo-EM reconstructions with sufficient resolution to visualise individual atoms in proteins has thus far been elusive. Here, we show that using a new electron source, energy filter and camera, we obtained a 1.7 Å resolution cryo-EM reconstruction for a prototypical human membrane protein, the β3 GABAA receptor homopentamer 3 . Such maps allow a detailed understanding of small molecule coordination, visualisation of solvent molecules and alternative conformations for multiple amino acids, as well as unambiguous building of ordered acidic side chains and glycans. Applied to mouse apo-ferritin, our strategy led to a 1.2 Å resolution .

Setting up and operating a cryo-EM laboratory

“…JEOL have their own in-column energy filter (the Omega filter), while TFS microscopes can be equipped with post-column filters built by Gatan or CEOS. Recently TFS also introduced their own filter (Nakane et al ., 2020).…”

“…In a very short time, cryo-electron microscopy (cryo-EM) has transformed from a technique practised by a small number of experts producing mostly medium-resolution structures with great effort, into a mainstream method for structural biologists, rivalling X-ray crystallography especially for samples that do not crystallize well, in particular, flexible and dynamic complexes and membrane proteins (Kühlbrandt, 2014; Bai et al ., 2015; Armache and Cheng, 2019). Electron microscopes, detectors and image processing tools are continuously improving, and at present, a suitable sample can reach a resolution up to 1.2 Å (Kato et al ., 2019; Nakane et al ., 2020; Pintilie et al ., 2020; Yip et al ., 2020). At the same time the size of suitable objects decreases and molecules down to 60 kDa can now be reconstructed (Khoshouei et al ., 2017; Herzik et al ., 2019).…”

Cryo‐EM of ABC transporters: an ice‐cold solution to everything?

“…A for an ideal test specimen [16,17], it is now demonstrated that cryo-EM has the potential to achieve sufficiently high resolutions for SBDD. However, the sample remains the limiting factor in terms of high resolution.…”

“…Owing to significant technological developments of hardware and software [9][10][11][12][13][14][15], cryo-EM is growing exponentially, and new breakthroughs in resolution and lowest analyzable molecule size are constantly witnessed [16][17][18]. The field is expanding not only by the amount of resolved structures, but also by the number of users and developers, as more and more researchers are turning to cryo-EM to answer their specific scientific questions.…”

TranSPHIRE: automated and feedback-optimized on-the-fly processing for cryo-EM

“…Consequently, automated processing especially facilitates the effective screening of many drug candidates. Recent advances in both hardware and software, which led to the first 1.2 Å resolution structures 6,7 , further demonstrate the potential of cryo-EM for drug discovery research in the near future.…”