Electron cryo-microscopy analyzes the structure of proteins and protein complexes in vitrified solution. Proteins tend to adsorb to the air-water interface in unsupported films of aqueous solution, which can result in partial or complete denaturation. We investigated the structure of yeast fatty acid synthase at the air-water interface by electron cryo-tomography and single-particle image processing. Around 90% of complexes adsorbed to the air-water interface are partly denatured. We show that the unfolded regions face the air-water interface. Denaturation by contact with air may happen at any stage of specimen preparation. Denaturation at the air-water interface is completely avoided when the complex is plunge-frozen on a substrate of hydrophilized graphene.

Balanced fusion and fission are key for proper function and physiology of mitochondria 1,2 . Remodelling of the mitochondrial inner membrane (IM) is mediated by dynamin-like Mitochondrial genome maintenance 1 protein (Mgm1) in fungi or the related Optic atrophy protein 1 (OPA1) in animals [3][4][5] . Mgm1 is required for the preservation of mitochondrial DNA in yeast 6 , whereas mutations in the OPA1 gene in humans are a common cause for autosomal dominant optic atrophy, a genetic disorder affecting the optical nerve 7,8 . Mgm1 and OPA1 are present in mitochondria as a membrane-integral long (l) form and a short (s) form that is soluble in the intermembrane space. Yeast strains expressing temperaturesensitive mutants of Mgm1 9,10 or mammalian cells devoid of OPA1 display fragmented mitochondria 11,12 , suggesting an important role of Mgm1/OPA1 in IM fusion. Consistently, only the mitochondrial outer membrane (OM), but not the IM, fuses in the absence of functional Mgm1 13,14 . Mgm1 and OPA1 have also been shown to maintain proper cristae architecture 10,14 . For example, OPA1 prevents the release of pro-apoptotic factors by tightening cristae junctions 15 . Finally, s-OPA1 localises to mitochondrial constriction sites, where it presumably promotes mitochondrial fission 16 . How Mgm1/OPA1 perform their diverse functions in membrane fusion, scission, and cristae organisation is at present unknown. Here, we present crystal and electron cryo-tomography (cryo-ET) structures of Chaetomium thermophilum Mgm1. Mgm1 consists of a GTPase domain, a bundle signalling element (BSE) domain, a stalk, and a paddle domain containing a membrane binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates assembly of bent tetramers into helical filaments. Cryo-ET of Mgm1-decorated lipid tubes and fluorescence microscopy experiments on reconstituted membrane tubes indicate how the tetramers assemble on positively or negatively curved membranes. Our findings convey how Mgm1/OPA1 filaments dynamically remodel the mitochondrial IM.We purified and crystallised a truncated s-Mgm1 isoform from the thermophilic fungus Chaetomium thermophilum (from here on Mgm1) (Fig. 1a, Extended Data Fig. 1a, Supplementary Data Fig. 1). Crystals of this construct grown in the absence of nucleotides diffracted to 3.6 Å resolution. The structure was solved by single anomalous dispersion (Extended Data Fig. 1b, c, Extended Data Table 1).The structure of Mgm1 contains four domains: A G domain, a bundle signalling element (BSE) domain, a stalk, and a paddle (Fig. 1a, b). The G domain closely resembles that of human dynamin (Extended Data Fig. 2). An interface across the nucleotide-binding site responsible for G domain dimerisation in the dynamin superfamily (the 'G interface') is highly conserved in Mgm1 (Extended Data Fig. 1e). The adjacent BSE domain consists of three helices derived from different regions of Mgm1 (Fig. 1a, b). The BSE domain contacts the G domain, as in the closed conformation of dynamin [17][18][19] . The M...

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.

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.

Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT. As for actinoporins, it has been proposed that the presence of cholesterol (Chol) and the coexistence of lipid phases increase binding to the target membrane and pore-forming ability. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, and the presence of lipid domains) on the activity of actinoporins or which regions of the membrane are the most favorable for protein insertion, oligomerization, and eventually pore formation. To gain insight into the role of membrane properties on the functional activity of St I, we studied its binding to monolayers and vesicles of phosphatidylcholine (PC), sphingomyelin (SM), and sterols inducing (ergosterol -Erg and cholesterol -Chol) or not (cholestenone - Cln) membrane phase segregation in liquid ordered (Lo) and liquid disordered (Ld) domains. This study revealed that St I binds and permeabilizes with higher efficiency sterol-containing membranes independently of their ability to form domains. We discuss the results in terms of the relevance of different membrane properties for the actinoporins mechanism of action, namely, molecular heterogeneity, specially potentiated in membranes with sterols inducers of phase separation (Chol or Erg) or Cln, a sterol noninducer of phase separation but with a high propensity to induce nonlamellar phase. The role of the Ld phase is pointed out as the most suitable platform for pore formation. In this regard, such regions in Chol-containing membranes seem to be the most favored due to its increased fluidity; this property promotes toxin insertion, diffusion, and oligomerization leading to pore formation.

Secretins form multimeric channels across the outer membrane of Gram-negative bacteria that mediate the import or export of substrates and/or extrusion of type IV pili. The secretin complex of Thermus thermophilus is an oligomer of the 757-residue PilQ protein, essential for DNA uptake and pilus extrusion. Here, we present the cryo-EM structure of this bifunctional complex at a resolution of ~7 Å using a new reconstruction protocol. Thirteen protomers form a large periplasmic domain of six stacked rings and a secretin domain in the outer membrane. A homology model of the PilQ protein was fitted into the cryo-EM map. A crown-like structure outside the outer membrane capping the secretin was found not to be part of PilQ. Mutations in the secretin domain disrupted the crown and abolished DNA uptake, suggesting a central role of the crown in natural transformation.

Since 1989, we have been involved in the development of a vaccine against Haemophilus influenzae type b. The new vaccine is based on the conjugation of synthetic oligosaccharides to tetanus toxoid. Our main goals have been (i) to verify the feasibility of using the synthetic antigen and (ii) to search for new production alternatives for this important infant vaccine. Overall, eight trials have already been conducted with adults, children (4 to 5 years old), and infants. We have described herein the details from the first two phase I clinical trials conducted with human adult volunteers under double blind, randomized conditions. The participants each received a single intramuscular injection to evaluate safety and initial immunogenicity. We have found an excellent safety profile and an antibody response similar to the one observed for the control vaccine.

Nitrate is an abundant nutrient and electron acceptor throughout Earth’s biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein’s electron transport chain, and elucidate the electron transfer pathways within the complex.