Streamlined structure determination by cryo-electron tomography and subtomogram averaging using TomoBEAR.

Structures of macromolecules in their native state provide unique unambiguous insights into their functions. Cryo-electron tomography combined with subtomogram averaging demonstrated the power to solve such structures in situ at resolutions in the range of 3 Angstrom for some macromolecules. In order to be applicable to the structural determination of the majority of macromolecules observable in cells in limited amounts, processing of tomographic data has to be performed in a high-throughput manner. Here we present TomoBEAR-a modular configurable workflow engine for streamlined processing of cryo-electron tomographic data for subtomogram averaging. TomoBEAR combines commonly used cryo-EM packages with reasonable presets to provide a transparent ("white box") approach for data management and processing. We demonstrate applications of TomoBEAR to two data sets of purified macromolecular targets, to an ion channel RyR1 in a membrane, and the tomograms of plasma FIB-milled lamellae and demonstrate the ability to produce high-resolution structures. TomoBEAR speeds up data processing, minimizes human interventions, and will help accelerate the adoption of in situ structural biology by cryo-ET. The source code and the documentation are freely available.

Simulation of Metal-Supported Metal-Nanoislands: A Comparison of DFT Methods.

We have evaluated various density functional theory (DFT) methods to simulate geometric, energetic, electronic, and hydrogen adsorption properties of metal-nanoparticles supported on metal surfaces. We used Pt and Pd nanoislands on Au(111) as model systems. The evaluated DFT methods include GGA (PW91, PBE, RPBE, revPBE, and PBESol), GGA with van der Waals (vdW) corrected (PBE-D3), GGA with optimized vdW functionals (revPBE-vdW), meta-GGA (SCAN and MS2), and the machine learning-based method BEEF-vdW. The results show that the various DFT methods yield similar geometric and electronic properties for Pt (or Pd) nanoislands on Au(111). The DFT methods also produce similar relative energetics for small Pt (or Pd) clusters with different conformations on Au(111). The results show that a triatomic cluster of Pt on Au(111) is more stable with a linear conformation. In contrast, a triatomic cluster of Pd is more stable with a triangular conformation. For clusters with four or more atoms, Pt and Pd clusters on Au(111) prefer non-linear conformation. We found that the various DFT methods yield different results only for the adsorption energy of hydrogen.

Asymmetric opening of the homopentameric 5-HT3A serotonin receptor in lipid bilayers.

Pentameric ligand-gated ion channels (pLGICs) of the Cys-loop receptor family are key players in fast signal transduction throughout the nervous system. They have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. We report three structures of the Cys-loop 5-HT3A serotonin receptor (5HT3R) reconstituted into saposin-based lipid bilayer discs: a symmetric and an asymmetric apo state, and an asymmetric agonist-bound state. In comparison to previously published 5HT3R conformations in detergent, the lipid bilayer stabilises the receptor in a more tightly packed, 'coupled' state, involving a cluster of highly conserved residues. In consequence, the agonist-bound receptor conformation adopts a wide-open pore capable of conducting sodium ions in unbiased molecular dynamics (MD) simulations. Taken together, we provide a structural basis for the modulation of 5HT3R by the membrane environment, and a model for asymmetric activation of the receptor.

Subnanometer-resolution structure determination in situ by hybrid subtomogram averaging - single particle cryo-EM.

Cryo-electron tomography combined with subtomogram averaging (StA) has yielded high-resolution structures of macromolecules in their native context. However, high-resolution StA is not commonplace due to beam-induced sample drift, images with poor signal-to-noise ratios (SNR), challenges in CTF correction, and limited particle number. Here we address these issues by collecting tilt series with a higher electron dose at the zero-degree tilt. Particles of interest are then located within reconstructed tomograms, processed by conventional StA, and then re-extracted from the high-dose images in 2D. Single particle analysis tools are then applied to refine the 2D particle alignment and generate a reconstruction. Use of our hybrid StA (hStA) workflow improved the resolution for tobacco mosaic virus from 7.2 to 4.4 Å and for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. These resolution gains make hStA a promising approach for other StA projects aimed at achieving subnanometer resolution.