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 reconstruction that, for the first time, offers a genuine atomic resolution view of a protein molecule using single particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualised in difference maps, allowing a direct analysis of hydrogen bonding networks. Combination of the technological advances described here with further approaches to accelerate data acquisition and improve sample quality provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery. Adrian Koh, Toby Darling and Jake Grimmett for support with computing; Garbi Lezcano Singla, Erik Franken for support with the EER format; and Gerald van Hoften and Gerard Hosmar for support with the Falcon-4 camera.

Routine sub-2.5 Å cryo-EM structure determination of B-family G protein-coupled receptors

“…Such contribution is also supported by the perceptibly better contrast in ZLF images (Figures 1E versus 1F). Our results recapitulate the improvement observed in a recent sub-2 Å cryo-EM reconstruction of a homopentameric GABA A receptor (Nakane et al, 2020). However, unlike the results in that study, we did not detect an improvement in the B-factor, possibly due to the wider energy limiting slit of 25 eV in our experiments compared to 5 eV in the GABAAR case.…”

“…In general, each of these settings may have a small and seemingly negligible effect on the result, but combining several optimal conditions has a cumulative effect and could lead to a noticeable improvement in map resolution and quality. As demonstrated here, and by recent atomic resolution structures (Nakane et al, 2020;Yip et al, 2020), fine-tuning of the experiment is crucial for pushing the performance limits of cryo-EM.…”

Adeno-Associated Virus (AAV-DJ)—Cryo-EM Structure at 1.56 Å Resolution

“…Approximately the same number of particles (47,000; 2.6 × 10 6 asymmetric units) were used as in an earlier (2017) 2.8 Å structure determination [ 22 ], in other words, this is a standard data collection regime without extraordinary efforts to maximize the resolution. The results reported here have not depended on the very latest microscope developments that have supported apoferritin structures beyond 1.5 Å [ 48 , 49 ].…”

“…By FSC 0.143 gold standard [ 47 ], the resolution is estimated to be 1.56 Å ( Figure 1 , Table 1 ). It is only recently with pre-print reports of apo-ferritin assemblies that this resolution has been exceeded for cryo-TEM of biomolecular assemblies [ 48 , 49 ]. There have been many measures of resolution in cryo-EM [ 50 ], and with the technological advances in recent years, there is growing appreciation of molecular features resolvable at the more common circa 3 Å resolution, we are in unchartered territory near 1.5 Å.…”

“…Their example is demonstrating that for other samples, instrumentation need not be limiting. Indeed the structure of a pentameric membrane protein has recently been reported at 1.7 Å resolution [ 48 ].…”