MotionCor2 software corrects for beam-induced sample motion, improving the resolution of cryo-EM reconstructions.

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MotionCor2: Anisotropic Correction of Beam-Induced Motion for Improved Cryo-Electron Microscopy

Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 A compared to previous RELION versions.

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New tools for automated high-resolution cryo-EM structure determination in RELION-3.

Diffraction (X-ray, neutron and electron) and electron cryo-microscopy are powerful methods to determine three-dimensional macromolecular structures, which are required to understand biological processes and to develop new therapeutics against diseases. The overall structure-solution workflow is similar for these techniques, but nuances exist because the properties of the reduced experimental data are different. Software tools for structure determination should therefore be tailored for each method. Phenix is a comprehensive software package for macromolecular structure determination that handles data from any of these techniques. Tasks performed with Phenix include data-quality assessment, map improvement, model building, the validation/rebuilding/refinement cycle and deposition. Each tool caters to the type of experimental data. The design of Phenix emphasizes the automation of procedures, where possible, to minimize repetitive and time-consuming manual tasks, while default parameters are chosen to encourage best practice. A graphical user interface provides access to many command-line features of Phenix and streamlines the transition between programs, project tracking and re-running of previous tasks.

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Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix

Gctf: Real-time CTF determination and correction

Transient receptor potential (TRP) channels are sensors for a wide range of cellular and environmental signals, but elucidating how these channels respond to physical and chemical stimuli has been hampered by a lack of detailed structural information. Here we exploit advances in electron cryo-microscopy to determine the structure of a mammalian TRP channel, TRPV1, at 3.4 A resolution, breaking the side-chain resolution barrier for membrane proteins without crystallization. Like voltage-gated channels, TRPV1 exhibits four-fold symmetry around a central ion pathway formed by transmembrane segments 5–6 (S5–S6) and the intervening pore loop, which is flanked by S1–S4 voltage-sensor-like domains. TRPV1 has a wide extracellular ‘mouth’ with a short selectivity filter. The conserved ‘TRP domain’ interacts with the S4–S5 linker, consistent with its contribution to allosteric modulation. Subunit organization is facilitated by interactions among cytoplasmic domains, including amino-terminal ankyrin repeats. These observations provide a structural blueprint for understanding unique aspects of TRP channel function. A high-resolution electron cryo-microscopy structure of the rat transient receptor potential (TRP) channel TRPV1 in its ‘closed’ state is presented; the overall structure of this ion channel is found to share some common features with voltage-gated ion channels, although several unique, TRP-specific features are also characterized. Transient receptor potential (TRP) channels are sensors for a wide range of physical and chemical stimuli. In the first of a pair of related papers, Maofu Liao et al. solve the high-resolution electron cryo-microscopy structure of rat TRPV1, the receptor for capsaicin (a pungent agent from chili peppers), in a 'closed' state. The overall structure is fairly similar to that of a voltage-gated ion channel, but there are several structural features unique to TRP channels. In the second paper, Erhu Cao et al. present the structures of rat TRPV1 in the presence of a peptide neurotoxin (resiniferatoxin) and in the presence of capsaicin, yielding structures of activated states of the channel. Comparison of the closed and open structures suggests that TRPV1 has a unique two-gate mechanism of channel activation.

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Structure of the TRPV1 ion channel determined by electron cryo-microscopy

In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM) has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electron-counting detector, we confirmed that electron beam-induced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to ∼3 A). Using this approach, we determined a 3.3-A-resolution structure of an ∼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency-key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.

Paul Mooney

Papers

Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM