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 .

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.

Arrestins regulate the signaling of ligand-activated, phosphorylated G-protein-coupled receptors (GPCRs). Different patterns of receptor phosphorylation (phosphorylation barcode) can modulate arrestin conformations, resulting in distinct functional outcomes (for example, desensitization, internalization, and downstream signaling). However, the mechanism of arrestin activation and how distinct receptor phosphorylation patterns could induce different conformational changes on arrestin are not fully understood. We analyzed how each arrestin amino acid contributes to its different conformational states. We identified a conserved structural motif that restricts the mobility of the arrestin finger loop in the inactive state and appears to be regulated by receptor phosphorylation. Distal and proximal receptor phosphorylation sites appear to selectively engage with distinct arrestin structural motifs (that is, micro-locks) to induce different arrestin conformations. These observations suggest a model in which different phosphorylation patterns of the GPCR C terminus can combinatorially modulate the conformation of the finger loop and other phosphorylation-sensitive structural elements to drive distinct arrestin conformation and functional outcomes.

Nanobodies (Nbs) are popular and versatile tools for structural biology because they have a compact single immunoglobulin domain organization. Nbs bind their target proteins with high affinities while reducing their conformational heterogeneity, and they stabilize multi-protein complexes. Here we demonstrate that engineered Nbs can also help overcome two major obstacles that limit the resolution of single-particle cryo-EM reconstructions: particle size and preferential orientation at the water-air interface. We have developed and characterised novel constructs, termed megabodies, by grafting Nbs into selected protein scaffolds to increase their molecular weight while retaining the full antigen binding specificity and affinity. We show that the megabody design principles are applicable to different scaffold proteins and recognition domains of compatible geometries and are amenable for efficient selection from yeast display libraries. Moreover, we used a megabody to solve the 2.5 Å resolution cryo-EM structure of a membrane protein that suffers from severe preferential orientation, the human GABA A b3 homopentameric receptor bound to its small-molecule agonist histamine.

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