Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting.

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.

AFM-based force spectroscopy unravels stepwise formation of the DNA transposition complex in the widespread Tn3 family mobile genetic elements.

Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recent data on the structural architecture of the transposition complex, the molecular mechanisms underlying the replicative transposition of these elements are still poorly understood. Here, we use force-distance curve-based atomic force microscopy to probe the binding of the TnpA transposase of Tn4430 to DNA molecules containing one or two transposon ends and to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathway for transposition complex formation and activation during which TnpA first binds as a dimer to a single transposon end and then undergoes a structural transition that enables it to bind the second end cooperatively and to become activated for transposition catalysis, the latter step occurring at a much faster rate for the TnpA mutants. Our study thus provides an unprecedented approach to probe the dynamic of a complex DNA processing machinery at the single-particle level.

Architecture and conformational switch mechanism of the ryanodine receptor.

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.

Mechanism of Tc toxin action revealed in molecular detail.

Tripartite Tc toxin complexes of bacterial pathogens perforate the host membrane and translocate toxic enzymes into the host cell, including in humans. The underlying mechanism is complex but poorly understood. Here we report the first, to our knowledge, high-resolution structures of a TcA subunit in its prepore and pore state and of a complete 1.7 megadalton Tc complex. The structures reveal that, in addition to a translocation channel, TcA forms four receptor-binding sites and a neuraminidase-like region, which are important for its host specificity. pH-induced opening of the shell releases an entropic spring that drives the injection of the TcA channel into the membrane. Binding of TcB/TcC to TcA opens a gate formed by a six-bladed ß-propeller and results in a continuous protein translocation channel, whose architecture and properties suggest a novel mode of protein unfolding and translocation. Our results allow us to understand key steps of infections involving Tc toxins at the molecular level.

An intrinsically disordered entropic switch determines allostery in Phd-Doc regulation.

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.

Reversible FMN dissociation from Escherichia coli respiratory complex I.

Respiratory complex I transfers electrons from NADH to quinone, utilizing the reaction energy to translocate protons across the membrane. It is a key enzyme of the respiratory chain of many prokaryotic and most eukaryotic organisms. The reversible NADH oxidation reaction is facilitated in complex I by non-covalently bound flavin mononucleotide (FMN). Here we report that the catalytic activity of E. coli complex I with artificial electron acceptors potassium ferricyanide (FeCy) and hexaamineruthenium (HAR) is significantly inhibited in the enzyme pre-reduced by NADH. Further, we demonstrate that the inhibition is caused by reversible dissociation of FMN. The binding constant (Kd) for FMN increases from the femto- or picomolar range in oxidized complex I to the nanomolar range in the NADH reduced enzyme, with an FMN dissociation time constant of ~5s. The oxidation state of complex I, rather than that of FMN, proved critical to the dissociation. Such dissociation is not observed with the T. thermophilus enzyme and our analysis suggests that the difference may be due to the unusually high redox potential of Fe-S cluster N1a in E. coli. It is possible that the enzyme attenuates ROS production in vivo by releasing FMN under highly reducing conditions.

Structure of the membrane domain of respiratory complex I.

Complex I is the first and largest enzyme of the respiratory chain, coupling electron transfer between NADH and ubiquinone to the translocation of four protons across the membrane. It has a central role in cellular energy production and has been implicated in many human neurodegenerative diseases. The L-shaped enzyme consists of hydrophilic and membrane domains. Previously, we determined the structure of the hydrophilic domain. Here we report the crystal structure of the Esherichia coli complex I membrane domain at 3.0 Å resolution. It includes six subunits, NuoL, NuoM, NuoN, NuoA, NuoJ and NuoK, with 55 transmembrane helices. The fold of the homologous antiporter-like subunits L, M and N is novel, with two inverted structural repeats of five transmembrane helices arranged, unusually, face-to-back. Each repeat includes a discontinuous transmembrane helix and forms half of a channel across the membrane. A network of conserved polar residues connects the two half-channels, completing the proton translocation pathway. Unexpectedly, lysines rather than carboxylate residues act as the main elements of the proton pump in these subunits. The fourth probable proton-translocation channel is at the interface of subunits N, K, J and A. The structure indicates that proton translocation in complex I, uniquely, involves coordinated conformational changes in six symmetrical structural elements.

Structural Details of the Ryanodine Receptor Calcium Release Channel and Its Gating Mechanism.

Ryanodine receptors (RyRs) are large intracellular calcium release channels that play a crucial role in coupling excitation to contraction in both cardiac and skeletal muscle cells. In addition, they are expressed in other cell types where their function is less well understood. Hundreds of mutations in the different isoforms of RyR have been associated with inherited myopathies and cardiac arrhythmia disorders. The structure of these important drug targets remained elusive for a long time, despite decades of intensive research. In the recent years, a technical revolution in the field of single particle cryogenic electron microscopy (SP cryo-EM) allowed solving high-resolution structures of the skeletal and cardiac RyR isoforms. Together with the structures of individual domains solved by X-ray crystallography, this resulted in an unprecedented understanding of the structure, gating and regulation of these largest known ion channels. In this chapter we describe the recently solved high-resolution structures of RyRs, discuss molecular details of the channel gating, regulation and the disease mutations. Additionally, we highlight important questions that require further progress in structural studies of RyRs.

The architecture of respiratory complex I.

Complex I is the first enzyme of the respiratory chain and has a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation by an unknown mechanism. Dysfunction of complex I has been implicated in many human neurodegenerative diseases. We have determined the structure of its hydrophilic domain previously. Here, we report the alpha-helical structure of the membrane domain of complex I from Escherichia coli at 3.9 A resolution. The antiporter-like subunits NuoL/M/N each contain 14 conserved transmembrane (TM) helices. Two of them are discontinuous, as in some transporters. Unexpectedly, subunit NuoL also contains a 110-A long amphipathic alpha-helix, spanning almost the entire length of the domain. Furthermore, we have determined the structure of the entire complex I from Thermus thermophilus at 4.5 A resolution. The L-shaped assembly consists of the alpha-helical model for the membrane domain, with 63 TM helices, and the known structure of the hydrophilic domain. The architecture of the complex provides strong clues about the coupling mechanism: the conformational changes at the interface of the two main domains may drive the long amphipathic alpha-helix of NuoL in a piston-like motion, tilting nearby discontinuous TM helices, resulting in proton translocation.

Apo and Aß46-bound γ-secretase structures provide insights into amyloid-ß processing by the APH-1B isoform.

Deposition of amyloid-ß (Aß) peptides in the brain is a hallmark of Alzheimer's disease. Aßs are generated through sequential proteolysis of the amyloid precursor protein by the γ-secretase complexes (GSECs). Aß peptide length, modulated by the Presenilin (PSEN) and APH-1 subunits of GSEC, is critical for Alzheimer's pathogenesis. Despite high relevance, mechanistic understanding of the proteolysis of Aß, and its modulation by APH-1, remain incomplete. Here, we report cryo-EM structures of human GSEC (PSEN1/APH-1B) reconstituted into lipid nanodiscs in apo form and in complex with the intermediate Aß46 substrate without cross-linking. We find that three non-conserved and structurally divergent APH-1 regions establish contacts with PSEN1, and that substrate-binding induces concerted rearrangements in one of the identified PSEN1/APH-1 interfaces, providing structural basis for APH-1 allosteric-like effects. In addition, the GSEC-Aß46 structure reveals an interaction between Aß46 and loop 1PSEN1, and identifies three other H-bonding interactions that, according to functional validation, are required for substrate recognition and efficient sequential catalysis.

The coupling mechanism of respiratory complex I - a structural and evolutionary perspective.

Complex I is a key enzyme of the respiratory chain in many organisms. This multi-protein complex with an intricate evolutionary history originated from the unification of prebuilt modules of hydrogenases and transporters. Using recently determined crystallographic structures of complex I we reanalyzed evolutionarily related complexes that couple oxidoreduction to trans-membrane ion translocation. Our analysis points to the previously unnoticed structural homology of the electron input module of formate dehydrogenlyases and subunit NuoG of complex I. We also show that all related to complex I hydrogenases likely operate via a conformation driven mechanism with structural changes generated in the conserved coupling site located at the interface of subunits NuoB/D/H. The coupling apparently originated once in evolutionary history, together with subunit NuoH joining hydrogenase and transport modules. Analysis of quinone oxidoreduction properties and the structure of complex I allows us to suggest a fully reversible coupling mechanism. Our model predicts that: 1) proton access to the ketone groups of the bound quinone is rigorously controlled by the protein, 2) the negative electric charge of the anionic ubiquinol head group is a major driving force for conformational changes. This article is part of a Special Issue entitled: 17th European Bioenergetics Conference (EBEC 2012).

A long road towards the structure of respiratory complex I, a giant molecular proton pump.

Complex I (NADH:ubiquinone oxidoreductase) is central to cellular energy production, being the first and largest enzyme of the respiratory chain in mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the inner mitochondrial membrane and is involved in a wide range of human neurodegenerative disorders. Mammalian complex I is composed of 44 different subunits, whereas the 'minimal' bacterial version contains 14 highly conserved 'core' subunits. The L-shaped assembly consists of hydrophilic and membrane domains. We have determined all known atomic structures of complex I, starting from the hydrophilic domain of Thermus thermophilus enzyme (eight subunits, nine Fe-S clusters), followed by the membrane domains of the Escherichia coli (six subunits, 55 transmembrane helices) and T. thermophilus (seven subunits, 64 transmembrane helices) enzymes, and finally culminating in a recent crystal structure of the entire intact complex I from T. thermophilus (536 kDa, 16 subunits, nine Fe-S clusters, 64 transmembrane helices). The structure suggests an unusual and unique coupling mechanism via long-range conformational changes. Determination of the structure of the entire complex was possible only through this step-by-step approach, building on from smaller subcomplexes towards the entire assembly. Large membrane proteins are notoriously difficult to crystallize, and so various non-standard and sometimes counterintuitive approaches were employed in order to achieve crystal diffraction to high resolution and solve the structures. These steps, as well as the implications from the final structure, are discussed in the present review.

Sterol derivative binding to the orthosteric site causes conformational changes in an invertebrate Cys-loop receptor.

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.

Structure of Escherichia coli OmpF porin from lipidic mesophase.

Outer membrane protein F, a major component of the Escherichia coli outer membrane, was crystallized for the first time in lipidic mesophase of monoolein in novel space groups, P1 and H32. Due to ease of its purification and crystallization OmpF can be used as a benchmark protein for establishing membrane protein crystallization in meso, as a "membrane lyzozyme". The packing of porin trimers in the crystals of space group H32 is similar to natural outer membranes, providing the first high-resolution insight into the close to native packing of OmpF. Surprisingly, interaction between trimers is mediated exclusively by lipids, without direct protein-protein contacts. Multiple ordered lipids are observed and many of them occupy identical positions independently of the space group, identifying preferential interaction sites of lipid acyl chains. Presence of ordered aliphatic chains close to a positively charged area on the porin surface suggests a position for a lipopolysaccharide binding site on the surface of the major E. coli porins.

Structural insight into Tn3 family transposition mechanism.

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.

Structure of Escherichia coli respiratory complex I reconstituted into lipid nanodiscs reveals an uncoupled conformation.

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.

Coma-corrected rapid single-particle cryo-EM data collection on the CRYO ARM 300.

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.

Assessing the JEOL CRYO ARM 300 for high-throughput automated single-particle cryo-EM in a multiuser environment.

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.

Influence of Lipid Mimetics on Gating of Ryanodine Receptor.

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 closed state of RyR1. Our biochemical results correlate with available structural data and suggest optimal conditions for structural studies of RyR1 gating.

Lipid Nanodiscs as a Tool for High-Resolution Structure Determination of Membrane Proteins by Single-Particle Cryo-EM.

The "resolution revolution" in electron cryomicroscopy (cryo-EM) profoundly changed structural biology of membrane proteins. Near-atomic structures of medium size to large membrane protein complexes can now be determined without crystallization. This significantly accelerates structure determination and also the visualization of small bound ligands. There is an additional advantage: the structure of membrane proteins can now be studied in their native or nearly native lipid bilayer environment. A popular lipid bilayer mimetic are lipid nanodiscs, which have been thoroughly characterized and successfully utilized in multiple applications. Here, we provide a guide for using lipid nanodiscs as a tool for single-particle cryo-EM of membrane proteins. We discuss general methodological aspects and specific challenges of protein reconstitution into lipid nanodiscs and high-resolution structure determination of the nanodisc-embedded complexes. Furthermore, we describe in detail case studies of two successful applications of nanodiscs in cryo-EM, namely, the structure determination of the rabbit ryanodine receptor, RyR1, and the pore-forming TcdA1 toxin subunit from Photorhabdus luminescens. We discuss cryo-EM-specific hurdles concerning sample homogeneity, distribution of reconstituted particles in vitreous ice, and solutions to overcome them.