CTFFIND4: Fast and accurate defocus estimation from electron micrographs.

We have developed new open-source software called cisTEM (computational imaging system for transmission electron microscopy) for the processing of data for high-resolution electron cryo-microscopy and single-particle averaging. cisTEM features a graphical user interface that is used to submit jobs, monitor their progress, and display results. It implements a full processing pipeline including movie processing, image defocus determination, automatic particle picking, 2D classification, ab-initio 3D map generation from random parameters, 3D classification, and high-resolution refinement and reconstruction. Some of these steps implement newly-developed algorithms; others were adapted from previously published algorithms. The software is optimized to enable processing of typical datasets (2000 micrographs, 200 k - 300 k particles) on a high-end, CPU-based workstation in half a day or less, comparable to GPU-accelerated processing. Jobs can also be scheduled on large computer clusters using flexible run profiles that can be adapted for most computing environments. cisTEM is available for download from cistem.org.

/pdf/cistem-user-friendly-software-for-single-particle-image-u95bi1zoed.pdf

cisTEM, user-friendly software for single-particle image processing

The mammalian mitochondrial electron transport chain (ETC) includes complexes I‑IV, as well as the electron transporters ubiquinone and cytochrome c. There are two electron transport pathways in the ETC: Complex I/III/IV, with NADH as the substrate and complex II/III/IV, with succinic acid as the substrate. The electron flow is coupled with the generation of a proton gradient across the inner membrane and the energy accumulated in the proton gradient is used by complex V (ATP synthase) to produce ATP. The first part of this review briefly introduces the structure and function of complexes I‑IV and ATP synthase, including the specific electron transfer process in each complex. Some electrons are directly transferred to O2 to generate reactive oxygen species (ROS) in the ETC. The second part of this review discusses the sites of ROS generation in each ETC complex, including sites IF and IQ in complex I, site IIF in complex II and site IIIQo in complex III, and the physiological and pathological regulation of ROS. As signaling molecules, ROS play an important role in cell proliferation, hypoxia adaptation and cell fate determination, but excessive ROS can cause irreversible cell damage and even cell death. The occurrence and development of a number of diseases are closely related to ROS overproduction. Finally, proton leak and uncoupling proteins (UCPS) are discussed. Proton leak consists of basal proton leak and induced proton leak. Induced proton leak is precisely regulated and induced by UCPs. A total of five UCPs (UCP1‑5) have been identified in mammalian cells. UCP1 mainly plays a role in the maintenance of body temperature in a cold environment through non‑shivering thermogenesis. The core role of UCP2‑5 is to reduce oxidative stress under certain conditions, therefore exerting cytoprotective effects. All diseases involving oxidative stress are associated with UCPs.

/pdf/mitochondrial-electron-transport-chain-ros-generation-and-1ba3ps939u.pdf

Mitochondrial electron transport chain, ROS generation and uncoupling (Review)

Mitochondrial rhomboid parl regulates cytochrome c release during apoptosis via OPA1 dependent cristae remodeling

Metabolic reprograming is an emerging hallmark of tumor biology and an actively pursued opportunity in discovery of oncology drugs. Extensive efforts have focused on therapeutic targeting of glycolysis, whereas drugging mitochondrial oxidative phosphorylation (OXPHOS) has remained largely unexplored, partly owing to an incomplete understanding of tumor contexts in which OXPHOS is essential. Here, we report the discovery of IACS-010759, a clinical-grade small-molecule inhibitor of complex I of the mitochondrial electron transport chain. Treatment with IACS-010759 robustly inhibited proliferation and induced apoptosis in models of brain cancer and acute myeloid leukemia (AML) reliant on OXPHOS, likely owing to a combination of energy depletion and reduced aspartate production that leads to impaired nucleotide biosynthesis. In models of brain cancer and AML, tumor growth was potently inhibited in vivo following IACS-010759 treatment at well-tolerated doses. IACS-010759 is currently being evaluated in phase 1 clinical trials in relapsed/refractory AML and solid tumors.

/pdf/an-inhibitor-of-oxidative-phosphorylation-exploits-cancer-1ns5bxutq5.pdf

An inhibitor of oxidative phosphorylation exploits cancer vulnerability.

Complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in the cell, powers ATP synthesis in mammalian mitochondria by using the reducing potential of NADH to drive protons across the inner mitochondrial membrane. Mammalian complex I (ref. 1) contains 45 subunits, comprising 14 core subunits that house the catalytic machinery (and are conserved from bacteria to humans) and a mammalian-specific cohort of 31 supernumerary subunits. Knowledge of the structures and functions of the supernumerary subunits is fragmentary. Here we describe a 4.2-A resolution single-particle electron cryomicroscopy structure of complex I from Bos taurus. We have located and modelled all 45 subunits, including the 31 supernumerary subunits, to provide the entire structure of the mammalian complex. Computational sorting of the particles identified different structural classes, related by subtle domain movements, which reveal conformationally dynamic regions and match biochemical descriptions of the 'active-to-de-active' enzyme transition that occurs during hypoxia. Our structures therefore provide a foundation for understanding complex I assembly and the effects of mutations that cause clinically relevant complex I dysfunctions, give insights into the structural and functional roles of the supernumerary subunits and reveal new information on the mechanism and regulation of catalysis.

/pdf/structure-of-mammalian-respiratory-complex-i-498kpjp3t8.pdf

Structure of mammalian respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase) is essential for oxidative phosphorylation in mammalian mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the energy-transducing inner membrane, providing electrons for respiration and driving ATP synthesis. Mammalian complex I contains 44 different nuclear- and mitochondrial-encoded subunits, with a combined mass of 1 MDa. The 14 conserved ‘core’ subunits have been structurally defined in the minimal, bacterial complex, but the structures and arrangement of the 30 ‘supernumerary’ subunits are unknown. Here we describe a 5 A resolution structure of complex I from Bos taurus heart mitochondria, a close relative of the human enzyme, determined by single-particle electron cryo-microscopy. We present the structures of the mammalian core subunits that contain eight iron–sulphur clusters and 60 transmembrane helices, identify 18 supernumerary transmembrane helices, and assign and model 14 supernumerary subunits. Thus, we considerably advance knowledge of the structure of mammalian complex I and the architecture of its supernumerary ensemble around the core domains. Our structure provides insights into the roles of the supernumerary subunits in regulation, assembly and homeostasis, and a basis for understanding the effects of mutations that cause a diverse range of human diseases. Complex I is the first enzyme of the mitochondrial electron transport chain and it is essential for oxidative phosphorylation in mammalian mitochondria; here the electron cryo-microscopy structure of complex I from bovine heart mitochondria is reported, advancing knowledge of its structure in mammals. The first enzyme of the mitochondrial electron transport chain, complex I (NADH:ubiquinone oxidoreductase), couples electron transfer from NADH to ubiquinone with proton translocation across the inner mitochondrial membrane, leading to the synthesis of ATP. This manuscript reports the electron cryo-microscopy structure of complex I from bovine heart mitochondria at 5 A resolution. The mammalian enzyme is much larger than the previously published structures of complex I from lower organisms. The authors assign the structures of 28 of the 44 subunits — the 14 conserved (core) subunits and 14 mammalian-specific subunits.

Architecture of mammalian respiratory complex I

There has been enormous progress during the last few years in the determination of three-dimensional biological structures by single particle electron cryomicroscopy (cryoEM), allowing maps to be obtained with higher resolution and from fewer images than required previously. This is due principally to the introduction of a new type of direct electron detector that has 2- to 3-fold higher detective quantum efficiency than available previously, and to the improvement of the computational algorithms for image processing. In spite of the great strides that have been made, quantitative analysis shows that there are still significant gains to be made provided that the problems associated with image degradation can be solved, possibly by minimising beam-induced specimen movement and charge build up during imaging. If this can be achieved, it should be possible to obtain near atomic resolution structures of smaller single particles, using fewer images and resolving more conformational states than at present, thus realising the full potential of the method. The recent popularity of cryoEM for molecular structure determination also highlights the need for lower cost microscopes, so we encourage development of an inexpensive, 100 keV electron cryomicroscope with a high-brightness field emission gun to make the method accessible to individual groups or institutions that cannot afford the investment and running costs of a state-of-the-art 300 keV installation. A key requisite for successful high-resolution structure determination by cryoEM includes interpretation of images and optimising the biochemistry and grid preparation to obtain nicely distributed macromolecules of interest. We thus include in this review a gallery of cryoEM micrographs that shows illustrative examples of single particle images of large and small macromolecular complexes.

/pdf/single-particle-electron-cryomicroscopy-trends-issues-and-3zzmil2vae.pdf

Single particle electron cryomicroscopy: trends, issues and future perspective.

In reviewing the structures of membrane proteins determined up to the end of 2009, we present in words and pictures the most informative examples from each family. We group the structures together according to their function and architecture to provide an overview of the major principles and variations on the most common themes. The first structures, determined 20 years ago, were those of naturally abundant proteins with limited conformational variability, and each membrane protein structure determined was a major landmark. With the advent of complete genome sequences and efficient expression systems, there has been an explosion in the rate of membrane protein structure determination, with many classes represented. New structures are published every month and more than 150 unique membrane protein structures have been determined. This review analyses the reasons for this success, discusses the challenges that still lie ahead, and presents a concise summary of the key achievements with illustrated examples selected from each class.

/pdf/structures-of-membrane-proteins-1yxwz1f78g.pdf

Structures of membrane proteins

Rhomboids are intramembrane proteases that use a catalytic dyad of serine and histidine for proteolysis. They are conserved in both prokaryotes and eukaryotes and regulate cellular processes as diverse as intercellular signalling, parasitic invasion of host cells, and mitochondrial morphology. Their widespread biological significance and consequent medical potential provides a strong incentive to understand the mechanism of these unusual enzymes for identification of specific inhibitors. In this study, we describe the structure of Escherichia coli rhomboid GlpG covalently bound to a mechanism-based isocoumarin inhibitor. We identify the position of the oxyanion hole, and the S1- and S2′-binding subsites of GlpG, which are the key determinants of substrate specificity. The inhibitor-bound structure suggests that subtle structural change is sufficient for catalysis, as opposed to large changes proposed from previous structures of unliganded GlpG. Using bound inhibitor as a template, we present a model for substrate binding at the active site and biochemically test its validity. This study provides a foundation for a structural explanation of rhomboid specificity and mechanism, and for inhibitor design.

/pdf/the-structural-basis-for-catalysis-and-substrate-specificity-2imy1brx74.pdf

Kutti R. Vinothkumar

Papers

Structure of mammalian respiratory complex I

Architecture of mammalian respiratory complex I

Single particle electron cryomicroscopy: trends, issues and future perspective.

Structures of membrane proteins

The structural basis for catalysis and substrate specificity of a rhomboid protease