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Balanced fusion and fission are key for the proper function and physiology of mitochondria1,2. Remodelling of the mitochondrial inner membrane is mediated by the dynamin-like protein mitochondrial genome maintenance 1 (Mgm1) in fungi or the related protein optic atrophy 1 (OPA1) in animals3–5. Mgm1 is required for the preservation of mitochondrial DNA in yeast6, whereas mutations in the OPA1 gene in humans are a common cause of autosomal dominant optic atrophy—a genetic disorder that affects the optic nerve7,8. Mgm1 and OPA1 are present in mitochondria as a membrane-integral long form and a short form that is soluble in the intermembrane space. Yeast strains that express temperature-sensitive mutants of Mgm19,10 or mammalian cells that lack OPA1 display fragmented mitochondria11,12, which suggests that Mgm1 and OPA1 have an important role in inner-membrane fusion. Consistently, only the mitochondrial outer membrane—not the inner membrane—fuses in the absence of functional Mgm113. Mgm1 and OPA1 have also been shown to maintain proper cristae architecture10,14; for example, OPA1 prevents the release of pro-apoptotic factors by tightening crista junctions15. Finally, the short form of OPA1 localizes to mitochondrial constriction sites, where it presumably promotes mitochondrial fission16. How Mgm1 and OPA1 perform their diverse functions in membrane fusion, scission and cristae organization is at present unknown. Here we present crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum. Mgm1 consists of a GTPase (G) domain, a bundle signalling element domain, a stalk, and a paddle domain that contains a membrane-binding site. Biochemical and cell-based experiments demonstrate that the Mgm1 stalk mediates the assembly of bent tetramers into helical filaments. Electron cryo-tomography studies 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 and OPA1 filaments dynamically remodel the mitochondrial inner membrane. Crystal and electron cryo-tomography structures of Mgm1 from Chaetomium thermophilum reveal that Mgm1 forms bent tetramers, which further assemble into helical filaments on both positively and negatively curved membranes.

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 (5HT(3)R) 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 5HT(3)R 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 5HT(3)R by the membrane environment, and a model for asymmetric activation of the receptor. Pentameric ligand-gated ion channels (pLGICs) are key players in neurotransmission and have been shown to be modulated by the lipid environment, however the underlying mechanism is not well understood. Here, the authors report structures of the pLGIC 5-HT3A serotonin receptor reconstituted into lipid bilayer discs and reveal lipid-protein interactions as well as asymmetric activation of the homopentameric receptor.

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., The oxidoreductase (NXR) multiprotein complex is a key enzyme in the nitrogen cycle. A detailed structural and biochemical characterization of NXR from the anammox bacterium Kuenenia stuttgartiensis shows that this complex is a filament-forming protein that catalysers both nitrite oxidation and nitrate reduction, and elucidates the mechanisms governing complex assembly and function.

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., Combining cryo-electron tomography with subtomogram averaging (StA) allows the in situ structure determination of proteins and protein complexes. Here, the authors present the hybrid StA (hStA) workflow that combines the advantages of single particle cryo-EM and StA and consists of a tomographic data collection scheme and a data processing workflow and they demonstrate how hStA can improve the resolution using two examples: the ion channel RyR1 and tobacco mosaic virus.

Cryo electron tomography (cryo-ET) combined with subtomogram averaging (StA) enables structural determination of macromolecules in their native context. A few structures were reported by StA at resolution higher than 4.5 Å, however all of these are from viral structural proteins or vesicle coats. Reaching high resolution for a broader range of samples is uncommon due to beam-induced sample drift, poor signal-to-noise ratio (SNR) of images, challenges in CTF correction, limited number of particles. Here we propose a strategy to address these issues, which consists of a tomographic data collection scheme and a processing workflow. Tilt series are collected with higher electron dose at zero-degree tilt in order to increase SNR. Next, after performing StA conventionally, we extract 2D projections of the particles of interest from the higher SNR images and use the single particle analysis tools to refine the particle alignment and generate a reconstruction. We benchmarked our proposedhybrid StA (hStA)workflow and improved the resolution for tobacco mosaic virus from 7.2 to 5.2 Å and the resolution for the ion channel RyR1 in crowded native membranes from 12.9 to 9.1 Å. We demonstrate thathStAcan improve the resolution obtained by conventional StA and promises to be a useful tool for StA projects aiming at subnanometer resolution or higher.

Volume alignment is a computationally intensive task. In Subtomogram Averaging (StA) from electron cryotomograms (CryoET), thousands of subtomograms are aligned to a reference, which may take hours until days of computational time. CryoET datasets contain a limited number of noisy projections, with very low signal–to–until ratio (SNR). The noisy subtomograms are aligned to a reference using cross—correlation, an operation that can be optimized when working with limited angle tomograms (LAT), as they are sparse in Fourier space. We propose a projected cross–correlation (pCC) algorithm, a faster approach to computing the cross—correlation between a limited angle (sub)–tomogram and a given reference, and we use pCC to design a new procedure for volume alignment. pCC employs the projections to calculate the cross–correlation with lower computational complexity, as it works with a set 2D projections instead of volumes. With this, we propose the Substacks Averaging (SsA) method as an alternative to the conventional Subtomogram Averaging (StA). Our results on test data shows that SsA is considerably faster than the reference StA implementation: for 41 projections (k= 41) and N=200, the SsA is 35 times faster, and for N=320, is 150 times faster. Furthermore, SsA results in higher precision of alignment of subtomograms at different noise levels.

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.

The cross-correlation is a fundamental operation in signal processing, as it is a measure of similarity and a tool to find translations between signals. Its implementation in Fourier space is used for large datasets, as it is faster than the one in real space, however, it does not consider any special properties which signals may have, as is the case of Limited Angle Tomography. The Fourier space of limited angle tomograms, which are reconstructed from a reduced number of projections, has a large number of empty values. As a consequence, most operations needed to calculate the cross-correlation are executed on empty data. To address this issue, we propose the projected Cross Correlation (pCC) method, which calculates the cross-correlation between a reference and a limited angle tomogram more efficiently. To reduce the number of operations, pCC follows a project, cross-correlate, reconstruct process, instead of the typical reconstruct, cross-correlate process. Both methods are equivalent, but the proposed one has lower computational complexity and provides significant speedup for larger tomograms, as we confirm with our experiments. Additionally, we propose the usage of a \(l_1\) penalty on the cross-correlation to improve its sensitivity and its robustness to noise. Our experimental results show that the improvements are achieved with no significant additional computational cost.

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 of the protein. 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 are those facing 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.

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.

The median filter is a non-linear filter used for removal of Salt & Pepper noise from images, where each pixel of the image is replaced by the median of its surrounding elements, which is calculated by sorting the data. The complexity of the sorting algorithms used for the median filters are $O(n^2)$ or $O(n)$ , depending on the kernel size. These algorithms were formulated for scalar single processor computers, with few of them successfully adapted and implemented for computers with a parallel architecture. In this paper we greatly improve the results of our earlier work, in which by means of a novel sorting algorithm, based on the Complementary Cumulative Distribution function, with $O(n)$ computational complexity and a highly parallelable structure, we presented a 2D median filter that achieved $O(1)$ or $O(n)$ computational complexity, depending memory constraints. The improvements are twofold: we propose a trade-off between $O(1)$ complexity and $O(n)$ complexity in order to improve the overall throughput; additionally we make use of the Salt & Pepper noise model to improve the image reconstruction quality with a small performance impact. The proposed algorithm have been implemented in three parallel programming models: SIMD Intel, Multicore Intel with SIMD, and SIMT (CUDA), achieving a peak throughput of 27.0, 100.1 and 91.6 megapixels per second respectively.

The median filter is a non-linear filter used for removal of salt and pepper noise from images. Each pixel of the image is replaced by the median of its surrounding elements, the median value is calculated by sorting the data. The complexity of the sorting algorithms used on the median filters are O(n2) or O(n), depending on the kernel size. Those algorithms were formulated for scalar single processor computers, with few of them successfully adapted and implemented for computer with a parallel architecture. In this paper we present a novel sorting algorithm, with O(n) computational complexity and a highly parallelizable structure, based on the Complementary Cumulative Distribution Function. Furthermore, a 2D median filter based on our proposed sorting algorithm can achieve O(1) complexity. We have implemented our proposed algorithm in two parallel architectures: SIMD Intel and CUDA, which have a throughput of 12.8 and 35 ∼ 57 megapixels per second respectively.