Cryo-EM of a heterogeneous biochemical fraction elucidates multiple protein complexes from a multicellular thermophilic eukaryote

Biomolecular complexes and their interactions govern cellular structure and function. Understanding their architecture is a prerequisite for dissecting the cell's inner workings, but their higher-order assembly is often transient and challenging for structural analysis. Here, we performed cryo-EM on a single, highly heterogeneous biochemical fraction derived from Chaetomium thermophilum cell extracts to visualize the biomolecular content of the multicellular eukaryote. After cryo-EM single-particle image processing, results showed that a simultaneous three-dimensional structural characterization of multiple chemically diverse biomacromolecules is feasible. Namely, the thermophilic, eukaryotic complexes of (a) ATP citrate-lyase, (b) Hsp90, (c) 20S proteasome, (d) Hsp60 and (e) UDP-glucose pyrophosphorylase were characterized. In total, all five complexes have been structurally dissected in a thermophilic eukaryote in a total imaged sample area of 190.64 μm2, and two, in particular, 20S proteasome and Hsp60, exhibit side-chain resolution features. The C. thermophilum Hsp60 near-atomic model was resolved at 3.46 Å (FSC = 0.143) and shows a hinge-like conformational change of its equatorial domain, highly similar to the one previously shown for its bacterial orthologue, GroEL. This work demonstrates that cryo-EM of cell extracts will greatly accelerate the structural analysis of cellular complexes and provide unprecedented opportunities to annotate architectures of biomolecules in a holistic approach

Cryo-EM structure of the SEA complex

The SEA complex (SEAC) is a growth regulator that acts as a GTPase-activating protein (GAP) towards Gtr1, a Rag GTPase that relays nutrient status to the Target of Rapamycin Complex 1 (TORC1) in yeast 1 . Functionally, the SEAC has been divided into two subcomplexes: SEACIT, which has GAP activity and inhibits TORC1, and SEACAT, which regulates SEACIT 2 . This system is conserved in mammals: the GATOR complex, consisting of GATOR1 (SEACIT) and GATOR2 (SEACAT), transmits amino acid 3 and glucose 4 signals to mTORC1. Despite its importance, the structure of SEAC/GATOR, and thus molecular understanding of its function, is lacking. Here, we solve the cryo-EM structure of the native eight-subunit SEAC. The SEAC has a modular structure in which a COPII-like cage corresponding to SEACAT binds two flexible wings, which correspond to SEACIT. The wings are tethered to the core via Sea3, which forms part of both modules. The GAP mechanism of GATOR1 is conserved in SEACIT, and GAP activity is unaffected by SEACAT in vitro. In vivo, the wings are essential for recruitment of the SEAC to the vacuole, primarily via the EGO complex. Our results indicate that rather than being a direct inhibitor of SEACIT, SEACAT acts as a scaffold for the binding of TORC1 regulators.</p

2.7 Å cryo-EM structure of vitrified M. Musculus H-chain apoferritin from a compact 200 keV cryo-microscope

Here we present the structure of mouse H-chain apoferritin at 2.7 Å (FSC = 0.143) solved by single particle cryogenic electron microscopy (cryo-EM) using a 200 kV device, the Thermo Fisher Glacios®. This is a compact, two-lens illumination system with a constant power objective lens, without any energy filters or aberration correctors, often thought of as a "screening cryo-microscope". Coulomb potential maps reveal clear densities for main chain carbonyl oxygens, residue side chains (including alternative conformations) and bound solvent molecules. We used a quasi-crystallographic reciprocal space approach to fit model coordinates to the experimental cryo-EM map. We argue that the advantages offered by (a) the high electronic and mechanical stability of the microscope, (b) the high emission stability and low beam energy spread of the high brightness Field Emission Gun (X-FEG), (c) direct electron detection technology and (d) particle-based Contrast Transfer Function (CTF) refinement have contributed to achieving high resolution. Overall, we show that basic electron optical settings for automated cryo-electron microscopy imaging can be used to determine structures approaching atomic resolution

ChIP-seq analysis revealing the mH2A1.1, mH2A1.2 and mH2A2 binding patterns in MEFs and ESCs. A.

Summary and Tornado plots depicting the binding of mH2A1.1 (left panels), mH2A1.2 (middle panels) and mH2A2 (right panels), in MEFs (upper panels) and ESCs (lower panels). Signal is normalized as log2FC (IP signal/Input signal) and peaks were defined using SICER2. B. Venn diagrams depicting the mH2A individual variant targets of the 73 mH2AMET/EMT genes in MEFs and ESCs. Targets were defined using the broad peaks derived from peak-calling analysis with SICER2 and peaks were annotated to genes with GREAT tool (±10 kb from the TSS). mH2A1.1 and mH2A2 have the most targets in MEFs, whereas in ESCs mH2A1.1 is the primary variant with direct binding at the 73 mH2AMET/EMT gene loci. C. Heatmaps depicting comparative ChIPseq analysis of mH2A1.1, mH2A1.2 and mH2A2 variants bound to the 73 mH2AMET/EMT genes in MEFs and ESCs as indicated. The average mH2A binding was calculated either at the -5kb regulatory region upstream from TSS or at the gene bodies after RPKM normalization. D. Intersection of the data presented in Fig 2B and S3C Fig. Genes with direct binding of a mH2A-bearing nucleosome are depicted in yellow and genes with no significant mH2A binding are depicted in black. (TIF)</p

Cryo-Electron Microscopy Snapshots of Eukaryotic Membrane Proteins in Native Lipid-Bilayer Nanodiscs

New technologies for purifying membrane-bound protein complexes in combination with cryo-electron microscopy (EM) have recently allowed the exploration of such complexes under near-native conditions. In particular, polymer-encapsulated nanodiscs enable the study of membrane proteins at high resolution while retaining protein–protein and protein–lipid interactions within a lipid bilayer. However, this powerful technology has not been exploited to address the important question of how endogenousas opposed to overexpressedmembrane proteins are organized within a lipid environment. In this work, we demonstrate that biochemical enrichment protocols for native membrane–protein complexes from Chaetomium thermophilum in combination with polymer-based lipid-bilayer nanodiscs provide a substantial improvement in the quality of recovered endogenous membrane–protein complexes. Mass spectrometry results revealed ∼1123 proteins, while multiple 2D class averages and two 3D reconstructions from cryo-EM data furnished prominent structural signatures. This integrated methodological approach to enriching endogenous membrane–protein complexes provides unprecedented opportunities for a deeper understanding of eukaryotic membrane proteomes

Solubilization of artificial mitochondrial membranes by amphiphilic copolymers of different charge

Certain amphiphilic copolymers form lipid-bilayer nanodiscs from artificial and natural membranes, thereby rendering incorporated membrane proteins optimal for structural analysis. Recent studies have shown that the amphiphilicity of a copolymer strongly determines its solubilization efficiency. This is especially true for highly negatively charged membranes, which experience pronounced Coulombic repulsion with polyanionic polymers. Here, we present a systematic study on the solubilization of artificial multicomponent lipid vesicles that mimic inner mitochondrial membranes, which harbor essential membrane-protein complexes. In particular, we compared the lipid-solubilization efficiencies of established anionic with less densely charged or zwitterionic and even cationic copolymers in low- and high-salt concentrations. The nanodiscs formed under these conditions were characterized by dynamic light scattering and negative-stain electron microscopy, pointing to a bimodal distribution of nanodisc diameters with a considerable fraction of nanodiscs engaging in side-by-side interactions through their polymer rims. Overall, our results show that some recent, zwitterionic copolymers are best suited to solubilize negatively charged membranes at high ionic strengths even at low polymer/lipid ratios

Identification of 73 mH2A_MET/EMT genes.

A. Diagrammatic representation of the rational followed for the identification of the 73 mH2AMET/EMT genes. B. Venn Diagram depicting the number and the identity of the commonly and differentially affected expression of the mH2AMET/EMT genes following individual mH2A KDs in MEFs (left panel) and ESCs (right panel). Each Venn diagram was constructed using the differentially expressed genes (DEGs) defined with p-adjusted2FC>0.58, or log2FCC. Heatmap depicting the expression levels (z-score) of the 73 mH2AMET/EMT genes in MEFs and ESCs (n = 2). D. Reconstruction of a mH2A-regulated gene network safeguarding the mesenchymal cell identity. The mH2A-regulated gene network in MEFs (MSCN) was reconstructed from 63 out of the 73 mH2AMET/EMT genes. The nodes were placed and grouped according to their known predominant subcellular localization (GO cellular component data) and molecular function (as annotated on the figure). Each connection (line) represents putative interactions and/or links between the indicated nodes. The expression trajectory of individual genes (nodes) during cellular reprogramming is depicted as a line within each node (Day 0, Day 3, Day 6, Day 9 and ESCs), according to our previous data [8] and publicly available data [32]. The border color of each node depicts the role assigned to this gene product in MET or EMT (orange: genes related exclusively, or mostly to EMT/mesenchymal phenotype, green: genes related exclusively, or mostly to MET/epithelial phenotype and grey: genes related to both EMT/mesenchymal and MET/epithelial phenotypes.</p