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Multidrug efflux pumps actively expel a wide range of toxic substrates from bacteria and play a major role in drug resistance. Here authors show the in situ structure of the efflux pump AcrAB-TolC obtained by electron cryo-tomography and subtomogram averaging.

iExoKras^G12D are engineered exosomes for the delivery of siRNA targeting KRAS^G12D. Here the authors describe the results of a phase I trial of iExoKrasG12D in patients with metastatic pancreatic cancer, reporting safety and clinical activity, as well as immunological correlates informing on tumor immune microenvironment reprograming and future combination with immune checkpoint inhibitors.’

e2gmm uses a deep neural network with a Gaussian representation to resolve the compositional and conformational variability within biomolecules using cryo-EM data.

In this work the authors investigate two types of secretins in Escherichia coli, GspD_α and GspD_β, and report the Cryo-ET in situ structures of their key intermediate states of during the translocation process. Yielding a resolution ranging from 9 Å to 19 Å, the structures allow the identification of different membrane interaction patterns and ways of transitioning the peptidoglycan layer. These results suggest two distinct models for the membrane translocation of GspD_α and GspD_β and provide insights into the inner to outer membrane biogenesis of T2SS secretins.

Recent developments in cryo-electron microscopy have enabled structure determination of large protein complexes at almost atomic resolution. Wang et al.combine some of these technologies into an effective workflow, and demonstrate the protocol by solving the atomic structure of an icosahedral RNA virus.

A method based on maximum likelihood density modification, adapted from X-ray crystallography, improves cryo-EM maps.

DNA supercoiling strongly affects its metabolism. By electron cryo-tomography, biochemical assays and molecular dynamics simulations, here the authors show that supercoiled DNA minicircles adopt unique and wide distributions of three-dimensional conformations, many with disrupted base pairs.

Here authors report cryo-EM structures of IP₃R1 which provide atomic details of IP₃, Ca²⁺ and ATP binding. Molecular motions of key domains and sidechains were found to regulate ligand binding and gating, which are validated by functional assays.

Cocrystal and cryo-EM structures of Geobacillus kaustophilus glyQ and Bacillus subtilis glyQS T-box-tRNA complexes establish a universal mechanism of amino acid sensing on tRNAs and gene regulation by T-box riboswitches.

An integrated pipeline for processing cryo-ET data implemented in EMAN2 streamlines data processing to minimize human bias, and improves the quality and resolution of resulting macromolecular structures, both in vitro and in cells.

The structure of the yeast nuclear pore complex, determined at sub-nanometre precision using an integrative approach that combines a wide range of data, reveals details of its architecture, transport mechanism and evolutionary origins.

Group II chaperonins, such as TriC/CCT, have a build-in lid that can cover the folding chamber and functions in an analogous way to the GroES-like proteins used by their Group I counterparts. Structural and modeling data suggest an allosteric mechanism of TriC lid closure that differs from GroES–GroEL systems.

This study has determined the electron cryomicroscopy structure of the mammalian type 1 InsP₃ receptor in a ligand-free state at 4.7 Å resolution; although the central Ca²⁺-conduction pathway is similar to other ion channels, the unique architecture of the C-terminal domains of the tetrameric channel suggests that a distinctive allosteric mechanism underlies the activation of InsP₃ gating.

This report describes the outcomes of the Data Management Challenges in 3D Electron Microscopy workshop. Key topics discussed include data models, validation and raw-data archiving. The meeting participants agreed that the EMDataBank should take the lead in addressing these issues, and concrete action points were agreed upon that will have a substantial impact on the accessibility of three-dimensional EM data in biology and medicine.

Group II chaperonins are present in eukaryotes and archaea and are essential mediators of cellular protein folding. This process is critically dependent on the closure of a built-in lid, which is triggered by ATP hydrolysis, but the structural rearrangements and molecular events leading to lid closure are unknown. Using cryo-electron microscopy, the structures of an archaeal group II chaperonin in the open and closed states are now reported, providing details of this mechanism.

Newly synthesized proteins are targeted to the SecY protein-conducting channel for translocation across the membrane; here, cryo-electron microscopy structures of inactive and active ribosome–channel complexes are presented, revealing that ribosome binding does not result in major structural changes to transmembrane regions of the channel, and that stable channel opening requires loop insertion of the translocating nascent chain.

An algorithm and software tool automating the annotation of subcellular features in cryo-electron tomography data is presented.

Equipping an electron microscope with Zernike phase-contrast optics dramatically increases the contrast of the images. Dai et al. describe how to successfully apply this technology for the acquisition and analysis of electron tomograms.

Wang et al characterise platelets in a mouse model of acute myeloid leukemia (AML) using Cryo-electron tomography (Cryo-ET) and find that 23% of platelets in pre-leukemic cells exhibit abnormal, round mitochondria with unfolded cristae. They also detect reduced ATP levels and altered expression of metabolism-related genes, altogether suggesting that platelet alterations may serve as a biomarker for AML.