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Myosin-2 is essential for processes as diverse as cell division and muscle contraction. Dephosphorylation of its regulatory light chain promotes an inactive, ‘shutdown’ state with the filament-forming tail folded onto the two heads 1 , which prevents filament formation and inactivates the motors 2 . The mechanism by which this happens is unclear. Here we report a cryo-electron microscopy structure of shutdown smooth muscle myosin with a resolution of 6 Å in the head region. A pseudo-atomic model, obtained by flexible fitting of crystal structures into the density and molecular dynamics simulations, describes interaction interfaces at the atomic level. The N-terminal extension of one regulatory light chain interacts with the tail, and the other with the partner head, revealing how the regulatory light chains stabilize the shutdown state in different ways and how their phosphorylation would allow myosin activation. Additional interactions between the three segments of the coiled coil, the motor domains and the light chains stabilize the shutdown molecule. The structure of the lever in each head is competent to generate force upon activation. This shutdown structure is relevant to all isoforms of myosin-2 and provides a framework for understanding their disease-causing mutations. The structure of myosin-2 in the shutdown state reveals how the shutdown state is stabilized and how phosphorylation of light chains allows myosin to be activated.

Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4 Å reconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10 ms, providing greater temporal resolution than previously reported mix-and-spray approaches.

Roundworm parasite infections are a major cause of human and livestock disease worldwide and a threat to global food security. Disease control currently relies on anthelmintic drugs to which roundworms are becoming increasingly resistant. An alternative approach is control by vaccination and 'hidden antigens', components of the worm gut not encountered by the infected host, have been exploited to produce Barbervax, the first commercial vaccine for a gut dwelling nematode of any host. Here we present the structure of H-gal-GP, a hidden antigen from Haemonchus contortus, the Barber's Pole worm, and a major component of Barbervax. We demonstrate its novel architecture, subunit composition and topology, flexibility and heterogeneity using cryo-electron microscopy, mass spectrometry, and modelling. Importantly, we demonstrate that complexes with the same architecture are present in other Strongylid roundworm parasites including human hookworm. This suggests a common ancestry and the potential for development of a unified hidden antigen vaccine.

Hemoglobin (Hb) is a tetrameric noncovalent complex consisting of two α- and two β-globin chains each associated with a heme group. Its exact assembly pathway is a matter of debate. Disorders of hemoglobin are the most common inherited disorders and subsequently the molecule has been extensively studied. This work attempts to further elucidate the structural properties of the hemoglobin tetramer and its components. Gas-phase conformations of hemoglobin tetramers and their constituents were investigated by means of traveling-wave ion mobility mass spectrometry. Sickle (HbS) and normal (HbA) hemoglobin molecules were analyzed to determine whether conformational differences in their quaternary structure could be observed. Rotationally averaged collision cross sections were estimated for tetramer, dimer, apo-, and holo-monomers with reference to a protein standard with known cross sections. Estimates of cross section obtained for the tetramers were compared to values calculated from X-ray crystallographic structures. HbS was consistently estimated to have a larger cross section than that of HbA, comparable with values obtained from X-ray crystallographic structures. Nontetrameric species observed included apo- and holo- forms of α- and β-monomers and heterodimers; α- and β-monomers in both apo- and holo- forms were found to have similar cross sections, suggesting they maintain a similar fold in the gas phase in both the presence and the absence of heme. Heme-deficient dimer, observed in the spectrum when analyzing commercially prepared Hb, was not observed when analyzing fresh blood. This implies that holo-α-apo-β is not an essential intermediate within the Hb assembly pathway, as previously proposed. Traveling-wave ion mobility mass spectrometry was used to investigate gas-phase conformations of hemoglobin tetramers and their constituents. Structural properties were elucidated.

Many virus capsids undergo exquisitely choreographed maturation processes in their host cells to produce infectious virions, and these remain poorly understood. As a tool for studying virus maturation, we transiently expressed the capsid protein of the insect virus Nudaurelia capensis omega virus (NωV) in Nicotiana benthamiana and were able to purify both immature procapsids and mature capsids from infiltrated leaves by varying the expression time. Cryo-EM analysis of the plant-produced procapsids and mature capsids to 6.6 Å and 2.7 Å resolution, respectively, reveals that in addition to large scale rigid body motions, internal regions of the subunits are extensively remodelled during maturation, creating the active site required for autocatalytic cleavage and infectivity. The mature particles are biologically active in terms of their ability to lyse membranes and have a structure that is essentially identical to authentic virus. The ability to faithfully recapitulate and visualize a complex maturation process in plants, including the autocatalytic cleavage of the capsid protein, has revealed a ~30 Å translation-rotation of the subunits during maturation as well as conformational rearrangements in the N and C-terminal helical regions of each subunit.Castells-Graells et al. demonstrate the maturation of insect virus Nudaurelia capensis omega virus (NωV) capsids in a plant model. This shows plants are a useful tool in the study of eukaryotic viruses across Kingdoms, and they identify new features in the maturation of the NωV capsid.

The preference for singly charged ion formation by MALDI makes it a better choice than electrospray ionization for profiling mixtures of N -glycans. For structural analysis, fragmentation of negative ions often yields more informative spectra than fragmentation of positive ones but such ions are more difficult to produce from neutral glycans under MALDI conditions. This work investigates conditions for the formation of both positive and negative ions by MALDI from N -linked glycans released from glycoproteins and their subsequent MS/MS and ion mobility behaviour. 2,4,6-Trihydroxyacetophenone (THAP) doped with ammonium nitrate was found to give optimal ion yields in negative ion mode. Ammonium chloride or phosphate also yielded prominent adducts but anionic carbohydrates such as sulfated N -glycans tended to ionize preferentially. Carbohydrates adducted with all three adducts (phosphate, chloride, and nitrate) produced good negative ion CID spectra but those adducted with iodide and sulfate did not yield fragment ions although they gave stronger signals. Fragmentation paralleled that seen following electrospray ionization providing superior spectra than could be obtained by PSD on MALDI-TOF instruments or with ion traps. In addition, ion mobility drift times of the adducted glycans and the ability of this technique to separate isomers also mirrored those obtained following ESI sample introduction. Ion mobility also allowed profiles to be obtained from samples whose MALDI spectra showed no evidence of such ions allowing the technique to be used in conditions where sample amounts were limiting. The method was applied to N -glycans released from the recombinant human immunodeficiency virus glycoprotein, gp120.

The analysis of glycosylation from native biological sources is often frustrated by the low abundances of available material. Here, ion mobility combined with electrospray ionization mass spectrometry have been used to extract the spectra of N -glycans released with PNGase F from a serial titration of recombinantly expressed envelope glycoprotein, gp120, from the human immunodeficiency virus (HIV). Analysis was also performed on gp120 expressed in the α-mannosidase inhibitor, and in a matched mammalian cell line deficient in GlcNAc transferase I. Without ion mobility separation, ESI spectra frequently contained no observable ions from the glycans whereas ions from other compounds such as detergents and residual buffer salts were abundant. After ion mobility separation on a Waters T-wave ion mobility mass spectrometer, the N -glycans fell into a unique region of the ion mobility/ m/z plot allowing their profiles to be extracted with good signal:noise ratios. This method allowed N -glycan profiles to be extracted from crude incubation mixtures with no clean-up even in the presence of surfactants such as NP40. Furthermore, this technique allowed clear profiles to be obtained from sub-microgram amounts of glycoprotein. Glycan profiles were similar to those generated by MALDI-TOF MS although they were more susceptible to double charging and fragmentation. Structural analysis could be accomplished by MS/MS experiments in either positive or negative ion mode but negative ion mode gave the most informative spectra and provided a reliable approach to the analysis of glycans from small amounts of glycoprotein.

Cardiac myosin is the molecular motor that drives heart contraction, powered by the energy-source ATP and through its interaction with actin tracks. Direct modulators of cardiac myosin function are promising treatments for inherited heart disease and heart failure. The inherited heart disease hypertrophic cardiomyopathy affects 1 in 500 people and is the most common cause of sudden onset cardiac death in the young, causing approximately 12 deaths in the UK every week. Heart failure is an increasing worldwide public health issue with an estimated prevalence of 64 million people, a mortality rate of approximately 50% within five years of diagnosis, and an estimated cost to the economy of $346 billion. Current treatments improve heart function but not patient survival. Direct myosin modulators have the potential to do both. Yet, despite a range of pre-clinical studies, we still have very little idea as to how these modulators work at the molecular level, which hinders their clinical usage and development.Here we explore the effect of cardiac myosin modulators on myosin structure and dynamics by use of cryogenic electron microscopy and cross-linking mass spectrometry. We also explore the effectiveness of mavacamten as a myosin inhibitor in the presence of specific disease-causing mutations by in vitro motility assay.We show how the myosin modulators mavacamten, OM and aficamten affect myosin structure and dynamics and how their effectiveness maybe influenced by the presence of hypertrophic cardiomyopathy mutations.This work has the potential to identify patient populations that can benefit most from specific modulator treatment, to predict prospective adverse effects, and to inform on the design of improved myosin modulators with better long-term outcomes.

Most sudden cardiac deaths in young people arise from hypertrophic cardiomyopathy, a genetic disease of the heart muscle, with many causative mutations found in the molecular motor beta-cardiac myosin that drives contraction. Therapeutic intervention has until recently been limited to symptomatic relief or invasive procedures. However, small molecule modulators of cardiac myosin are promising therapeutic options to target disease progression. Mavacamten is the first example to gain FDA approval but its molecular mode of action remains unclear, limiting our understanding of its functional effects in disease. To better understand this, we solved the cryoEM structures of beta-cardiac heavy meromyosin in three ADP.Pi-bound states, the primed motor domain in the presence and absence of mavacamten, and the sequestered autoinhibited interacting-heads motif (IHM) in complex with mavacamten, to 2.9 Å, 3.4 Å and 3.7 Å global resolution respectively. Together with quantitative crosslinking mass spectrometric analysis, these structures reveal how mavacamten inhibits myosin. Mavacamten stabilises ADP.Pi binding, stalling the motor domain in a primed state, reducing motor dynamics required for actin-binding cleft closure, and slowing progression through the force generation cycle. Within the two-headed myosin molecule, these effects are propagated and lead to stabilisation of the IHM, through increased contacts at the motor-motor interface. Critically, while mavacamten treatment can thus rescue cardiac muscle relaxation in diastole, it can also reduce contractile output in systole in the heart.

Hemoglobin (Hb) is a tetrameric noncovalent complex consisting of two alpha- and two beta-globin chains each associated with a heme group. Its exact assembly pathway is a matter of debate. Disorders of hemoglobin are the most common inherited disorders and subsequently the molecule has been extensively studied. This work attempts to further elucidate the structural properties of the hemoglobin tetramer and its components. Gas-phase conformations of hemoglobin tetramers and their constituents were investigated by means of traveling-wave ion mobility mass spectrometry. Sickle (HbS) and normal (HbA) hemoglobin molecules were analyzed to determine whether conformational differences in their quaternary structure could be observed. Rotationally averaged collision cross sections were estimated for tetramer, dimer, apo-, and holo-monomers with reference to a protein standard with known cross sections. Estimates of cross section obtained for the tetramers were compared to values calculated from X-ray crystallographic structures. HbS was consistently estimated to have a larger cross section than that of HbA, comparable with values obtained from X-ray crystallographic structures. Nontetrameric species observed included apo- and holo- forms of alpha- and beta-monomers and heterodimers; alpha- and beta-monomers in both apo- and holo- forms were found to have similar cross sections, suggesting they maintain a similar fold in the gas phase in both the presence and the absence of heme. Heme-deficient dimer, observed in the spectrum when analyzing commercially prepared Hb, was not observed when analyzing fresh blood. This implies that holo-alpha-apo-beta is not an essential intermediate within the Hb assembly pathway, as previously proposed. Graphical Abstract Traveling-wave ion mobility mass spectrometry was used to investigate gas-phase conformations of hemoglobin tetramers and their constituents. Structural properties were elucidated. Full-size image (40K) High-quality image (351K)