SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) ...data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 Å structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.
Biogenesis and dynamics of thylakoid membranes likely involves membrane fusion events. Membrane attachment of the inner membrane-associated protein of 30 kDa (IM30) affects the structure of the lipid ...bilayer, finally resulting in membrane fusion. Yet, how IM30 triggers membrane fusion is largely unclear. IM30 monomers pre-assemble into stable tetrameric building blocks, which further align to form oligomeric ring structures, and differently sized IM30 rings bind to membranes. Based on a 3D reconstruction of IM30 rings, we locate the IM30 loop 2 region at the bottom of the ring and show intact membrane binding but missing fusogenic activity of loop 2 mutants. However, helix 7, which has recently been shown to mediate membrane binding, was located at the oppossite, top side of IM30 rings. We propose that a two-sided IM30 ring complex connects two opposing membranes, finally resulting in membrane fusion. Thus, IM30-mediated membrane fusion requires a Janus-faced IM30 ring.
Display omitted
•IM30 monomers assemble into stable tetrameric building blocks prior to ring formation•3D reconstructions of different IM30 rings are presented•Helix 7 and loop 2 are located at opposite sides of IM30 rings•IM30-mediated membrane fusion requires a Janus-faced IM30 ring
The IM30 protein is a membrane fusion protein that forms large oligomeric rings from tetrameric building blocks. Saur et al. present 3D reconstructions of the Janus-faced IM30 ring, which illustrates membrane binding properties as well as the membrane fusion activity of IM30.
This article proposes an enhanced version of the deadbeat flux vector controller (DBFC) as a one single control law that can operate in the entire torque-speed plane. The operation at any feasible ...modulation index can be accomplished by adequate determination of the flux trajectories at the different operating regions (e.g., PWM, overmodulation (I and II), and six-step). Continuous and seamless transition between the four operating regions is guaranteed, where the modulation index changes linearly with speed between PWM and six-step (without abrupt change in torque or acoustic problems). With the proposed strategy, undesirable torque dynamics, stability problems, and increased computational efforts associated with using multiple control laws are avoided. The transient performance of DBFC at the maximum voltage limit is analyzed in detail in the flux plane. A time-optimal torque control algorithm is developed to achieve the fastest possible torque dynamics and to considerably reduce the settling time, without the use of a voltage margin. The torque can be controlled with high accuracy and high robustness to machine parameter variations. With DBFC, no tradeoff between good steady state six-step behavior and good transient performance is needed due to the decoupling of switching and calculation frequencies. The proposed DBFC controller offers valuable features, and it is simple to implement. Simulation and experimental results are provided to validate the proposed control algorithm, which is implemented on an automotive microcontroller with a high-power/high-performance automotive traction machine.
Nicotinic acetylcholine receptors (nAChR) play important neurophysiological roles and are of considerable medical relevance. They have been studied extensively, greatly facilitated by the gastropod ...acetylcholine-binding proteins (AChBP) which represent soluble structural and functional homologues of the ligand-binding domain of nAChR. All these proteins are ring-like pentamers. Here we report that AChBP exists in the hemolymph of the planorbid snail Biomphalaria glabrata (vector of the schistosomiasis parasite) as a regular pentagonal dodecahedron, 22 nm in diameter (12 pentamers, 60 active sites). We sequenced and recombinantly expressed two ∼25 kDa polypeptides (BgAChBP1 and BgAChBP2) with a specific active site, N-glycan site and disulfide bridge variation. We also provide the exon/intron structures. Recombinant BgAChBP1 formed pentamers and dodecahedra, recombinant BgAChBP2 formed pentamers and probably disulfide-bridged di-pentamers, but not dodecahedra. Three-dimensional electron cryo-microscopy (3D-EM) yielded a 3D reconstruction of the dodecahedron with a resolution of 6 Å. Homology models of the pentamers docked to the 6 Å structure revealed opportunities for chemical bonding at the inter-pentamer interfaces. Definition of the ligand-binding pocket and the gating C-loop in the 6 Å structure suggests that 3D-EM might lead to the identification of functional states in the BgAChBP dodecahedron.
Fragment-based drug discovery using cryo-EM Saur, Michael; Hartshorn, Michael J.; Dong, Jing ...
Drug discovery today,
March 2020, 2020-03-00, Letnik:
25, Številka:
3
Journal Article
Recenzirano
Odprti dostop
•Cryo-EM maps can be used for structure-based drug design.•Cryo-EM can provide the throughput necessary to support fragment-based screening.•We present the first cryo-EM structure of the oncology ...target PKM2.•We discuss future developments that will enhance fragment-based drug discovery.
Recent advances in electron cryo-microscopy (cryo-EM) structure determination have pushed the resolutions obtainable by the method into the range widely considered to be of utility for drug discovery. Here, we review the use of cryo-EM in fragment-based drug discovery (FBDD) based on in-house method development. We demonstrate not only that cryo-EM can reveal details of the molecular interactions between fragments and a protein, but also that the current reproducibility, quality, and throughput are compatible with FBDD. We exemplify this using the test system β-galactosidase (Bgal) and the oncology target pyruvate kinase 2 (PKM2).
Through a combination of flux and current observers, deadbeat-direct torque and flux control for interior permanent magnet synchronous machines has preferred features comparing to the existing ...current vector control (CVC). These include simpler flux weakening control, less parameter sensitivity with increasing speed due to the Gopinath-style flux observer and evading of anti-windup strategies at the inverter voltage limit. The operation throughout the entire torque-speed range can be accomplished with a single control law. For the second flux weakening region, the algorithm inherently achieves the maximum torque per flux (MTPF) operation by applying the square-root-condition (SRC). In contrast to CVC, no look-up-table nor solving the parameter-dependent MTPF equation is required. In this article, the equivalence of the SRC method to the conventional MTPF strategy is demonstrated analytically, graphically, in simulation, and through experimental results without any stability problems. A sensitivity analysis regarding the machine parameters and iron losses compares and evaluates both methods. Superior performance of the SRC algorithm compared to the conventional method is proven.
Kinetochores are macromolecular assemblies that connect chromosomes to spindle microtubules (MTs) during mitosis. The metazoan-specific ≈800-kD ROD-Zwilch-ZW10 (RZZ) complex builds a fibrous corona ...that assembles on mitotic kinetochores before MT attachment to promote chromosome alignment and robust spindle assembly checkpoint signaling. In this study, we combine biochemical reconstitutions, single-particle electron cryomicroscopy, cross-linking mass spectrometry, and structural modeling to build a complete model of human RZZ. We find that RZZ is structurally related to self-assembling cytosolic coat scaffolds that mediate membrane cargo trafficking, including Clathrin, Sec13-Sec31, and αβ'ε-COP. We show that Spindly, a dynein adaptor, is related to BicD2 and binds RZZ directly in a farnesylation-dependent but membrane-independent manner. Through a targeted chemical biology approach, we identify ROD as the Spindly farnesyl receptor. Our results suggest that RZZ is dynein's cargo at human kinetochores.
The thylakoid membrane of chloroplasts and cyanobacteria is a unique internal membrane system harbouring the complexes of the photosynthetic electron transfer chain. Despite their apparent ...importance, little is known about the biogenesis and maintenance of thylakoid membranes. Although membrane fusion events are essential for the formation of thylakoid membranes, proteins involved in membrane fusion have yet to be identified in photosynthetic cells or organelles. Here we show that IM30, a conserved chloroplast and cyanobacterial protein of approximately 30 kDa binds as an oligomeric ring in a well-defined geometry specifically to membranes containing anionic lipids. Triggered by Mg(2+), membrane binding causes destabilization and eventually results in membrane fusion. We propose that IM30 establishes contacts between internal membrane sites and promotes fusion to enable regulated exchange of proteins and/or lipids in cyanobacteria and chloroplasts.
PDF
