Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid reaction initiation and plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a resolution of 2.1 Å. trEM experiments on GroEL:GroES chaperonin complex resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex.

Cys-loop receptors or pentameric ligand-gated ion channels are mediators of electrochemical signaling throughout the animal kingdom. Because of their critical function in neurotransmission and high potential as drug targets, Cys-loop receptors from humans and closely related organisms have been thoroughly investigated, whereas molecular mechanisms of neurotransmission in invertebrates are less understood. When compared with vertebrates, the invertebrate genomes underwent a drastic expansion in the number of the nACh-like genes associated with receptors of unknown function. Understanding this diversity contributes to better insight into the evolution and possible functional divergence of these receptors. In this work, we studied orphan receptor Alpo4 from an extreme thermophile worm Alvinella pompejana. Sequence analysis points towards its remote relation to characterized nACh receptors. We solved the cryo-EM structure of the lophotrochozoan nACh-like receptor in which a CHAPS molecule is tightly bound to the orthosteric site. We show that the binding of CHAPS leads to extending of the loop C at the orthosteric site and a quaternary twist between extracellular and transmembrane domains. Both the ligand binding site and the channel pore reveal unique features. These include a conserved Trp residue in loop B of the ligand binding site which is flipped into an apparent self-liganded state in the apo structure. The ion pore of Alpo4 is tightly constricted by a ring of methionines near the extracellular entryway of the channel pore. Our data provide a structural basis for a functional understanding of Alpo4 and hints towards new strategies for designing specific channel modulators.

Transposons are diverse mobile genetic elements that play the critical role as genome architects in all domains of life. Tn3 is a widespread family and among the first identified bacterial transposons famed for their contribution to the dissemination of antibiotic resistance. Transposition within this family is mediated by a large TnpA transposase, which facilitates both transposition and target immunity. Howtever, a structural framework required for understanding the mechanism of TnpA transposition is lacking. Here, we describe the cryo-EM structures of TnpA from Tn4430 in the apo form and paired with transposon ends before and after DNA cleavage and strand transfer. We show that TnpA has an unusual architecture and exhibits a family specific regulatory mechanism involving metamorphic refolding of the RNase H-like catalytic domain. The TnpA structure, constrained by a double dimerization interface, creates a peculiar topology that suggests a specific role for the target DNA in transpososome assembly and activation.

Respiratory complex I is a multi-subunit membrane protein complex that reversibly couples NADH oxidation and ubiquinone reduction with proton translocation against transmembrane potential. Complex I from Escherichia coli is among the best functionally characterized complexes, but its structure remains unknown, hindering further studies to understand the enzyme coupling mechanism. Here, we describe the single particle cryo-electron microscopy (cryo-EM) structure of the entire catalytically active E. coli complex I reconstituted into lipid nanodiscs. The structure of this mesophilic bacterial complex I displays highly dynamic connection between the peripheral and membrane domains. The peripheral domain assembly is stabilized by unique terminal extensions and an insertion loop. The membrane domain structure reveals novel dynamic features. Unusual conformation of the conserved interface between the peripheral and membrane domains suggests an uncoupled conformation of the complex. Considering constraints imposed by the structural data, we suggest a new simple hypothetical coupling mechanism for the molecular machine.

Single-particle cryogenic electron microscopy has recently become a major method for determining the structures of proteins and protein complexes. This has markedly increased the demand for throughput of high-resolution electron microscopes, which are required to produce high-resolution images at high rates. An increase in data-collection throughput can be achieved by using large beam-image shifts combined with off-axis coma correction, enabling the acquisition of multiple images from a large area of the EM grid without moving the microscope stage. Here, the optical properties of the JEOL CRYO ARM 300 electron microscope equipped with a K3 camera were characterized under off-axis illumination conditions. It is shown that efficient coma correction can be achieved for beam-image shifts with an amplitude of at least 10 µm, enabling a routine throughput for data collection of between 6000 and 9000 images per day. Use of the benchmark for the rapid data-collection procedure (with beam-image shifts of up to 7 µm) on apoferritin resulted in a reconstruction at a resolution of 1.7 Å. This demonstrates that the rapid automated acquisition of high-resolution micrographs is possible using a CRYO ARM 300.

Single-particle cryo-EM has become an indispensable structural biology method. It requires regular access to high-resolution electron cryogenic microscopes. To fully utilize the capacity of the expensive high-resolution instruments, the time used for data acquisition and the rate of data collection have to be maximized. This in turn requires high stability and high uptime of the instrument. One of the first 300 kV JEOL CRYO ARM 300 microscopes has been installed at the cryo-EM facility BECM at VIB-VUB, Brussels, where the microscope is used for continuous data collection on multiple projects. Here, the suitability and performance of the microscope is assessed for high-throughput single-particle data collection. In particular, the properties of the illumination system, the stage stability and ice contamination rates are reported. The microscope was benchmarked using mouse heavy-chain apoferritin which was reconstructed to a resolution of 1.9 Å. Finally, uptime and throughput statistics of the instrument accumulated during the first six months of the facility operation in user access mode are reported.

Understanding gating principles of ion channels at high resolution is of great importance. Here we investigate the conformational transition from closed to open state in ryanodine receptor 1 (RyR1) reconstituted into lipid nanodiscs. RyR1 is a homotetrameric giant ion channel that couples excitation of muscle cells to fast calcium release from the sarcoplasmic reticulum. Using single-particle cryo-EM we show that RyR1 reconstituted into lipid nanodiscs is stabilized in the open conformation when bound to the plant toxin ryanodine, but not in the presence of its physiological activators, calcium, and ATP. Further, using ryanodine binding assays we show that membrane mimetics influence RyR1 transition between closed and open-channel conformations. We find that all detergents, including fluorinated detergents, added to nanodiscs, stabilize the closed state of RyR1. Our biochemical results correlate with available structural data and suggest optimal conditions for structural studies of RyR1 gating.

Bacteria can become transiently tolerant to several classes of antibiotics. This phenomenon known as persistence is regulated by small genetic elements called toxin-antitoxin modules with intricate yet often poorly understood self-regulatory features. Here, we describe the structures of molecular complexes and interactions that drive the transcription regulation of the ccdAB toxin-antitoxin module. Low specificity and affinity of the antitoxin CcdA2 for individual binding sites on the operator are enhanced by the toxin CcdB2, which bridges the CcdA2 dimers. This results in a unique extended repressing complex that spirals around the operator and presents equally spaced DNA binding sites. The multivalency of binding sites induces a digital on-off switch for transcription, regulated by the toxin:antitoxin ratio. The ratio at which this switch occurs is modulated by non-specific interactions with the excess chromosomal DNA. Altogether, we present the molecular mechanisms underlying the ratio-dependent transcriptional regulation of the ccdAB operon.

Conditional cooperativity is a common mechanism involved in transcriptional regulation of prokaryotic type II toxin-antitoxin operons and is intricately related to bacterial persistence. It allows the toxin component of a toxin-antitoxin module to act as a co-repressor at low doses of toxin as compared to antitoxin. When toxin level exceeds a certain threshold, however, the toxin becomes a de-repressor. Most antitoxins contain an intrinsically disordered region (IDR) that typically is involved in toxin neutralization and repressor complex formation. To address how the antitoxin IDR is involved in transcription regulation, we studied the phd-doc operon from bacteriophage P1. We provide evidence that the IDR of Phd provides an entropic barrier precluding full operon repression in the absence of Doc. Binding of Doc results in a cooperativity switch and consequent strong operon repression, enabling context-specific modulation of the regulatory process. Variations of this theme are likely to be a common mechanism in the autoregulation of bacterial operons that involve intrinsically disordered regions.

Muscle contraction is initiated by the release of calcium (Ca(2+)) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins. Numerous mutations in RyRs have been associated with human diseases. The molecular mechanism underlying the complex regulation of RyRs is poorly understood. Using electron cryomicroscopy, here we determine the architecture of rabbit RyR1 at a resolution of 6.1 Å. We show that the cytoplasmic moiety of RyR1 contains two large α-solenoid domains and several smaller domains, with folds suggestive of participation in protein-protein interactions. The transmembrane domain represents a chimaera of voltage-gated sodium and pH-activated ion channels. We identify the calcium-binding EF-hand domain and show that it functions as a conformational switch allosterically gating the channel.