Biology subjects

White, H. D.

Publications and source records attributed to White, H. D..

4 recordsLinked to original sources

bioRxiv2026

The structure of the native cardiac crossbridge in the rigor state.

Cardiac contraction is driven by double-headed myosin cycling on cardiac thin filaments, where troponin-tropomyosin regulates myosin access to actin. Prior research used a single-headed myosin bound to bare actin, thereby limiting insight into coordination between myosin heads and the influence of troponin-tropomyosin on actomyosin interactions. Here, we report a high-resolution structure of the native cardiac rigor cross-bridge formed by heavy meromyosin bound to the thin filament. We show that direct communication between the two bound heads, uneven interactions between the heads and tropomyosin, and spatial constraints imposed by troponin govern myosin placement along the thin filament. Additionally, the two heads display non-equivalent motor-light chain interactions, yielding distinct lever-arm conformations indicative of asymmetric intramolecular strain. Together, these findings provide a structural framework for how the two myosin heads coordinate and how the components of the thin filament are integrated into force generation by active cross-bridges.

biophysics↗

bioRxiv2024

Swinging lever mechanism of myosin directly demonstrated by time-resolved cryoEM

Myosins are essential for producing force and movement in cells through their interactions with F-actin. Generation of movement is proposed to occur through structural changes within the myosin motor domain, fuelled by ATP hydrolysis, that are amplified by a lever swing1, transitioning myosin from a primed (pre-powerstroke) state to a post-powerstroke state. However, the initial, primed actomyosin state, proposed to form prior to lever swing, has never been observed. Nor has the mechanism by which actin catalyses myosin ATPase activity been resolved. To address this, we performed time-resolved cryoEM of a myosin-5 mutant having slow hydrolysis product release. Primed actomyosin was captured 10 ms after mixing primed myosin with F-actin, whereas post-powerstroke actomyosin predominated at 120 ms, with no abundant intermediate structures. The structures were solved to 4.4[A] and 4.2[A] global resolution respectively. The primed motor binds to actin through its lower 50 kDa subdomain, with the actin-binding cleft open and Pi release prohibited. N-terminal actin interactions with myosin promote rotation of the upper 50 kDa subdomain, which closes the actin-binding cleft, and enables Pi release. Formation of upper 50 kDa subdomain interactions with actin creates the strong-binding interface required for effective force production. The myosin-5 lever swings through an angle of 93{degrees}, predominantly along the actin axis, with little twisting, to produce the post-powerstroke state. The magnitude of the lever swing matches the typical step length of myosin-5 walking along actin. These time-resolved structures directly demonstrate the swinging lever mechanism, ending decades of conjecture on how myosin produces force and movement.

biochemistry↗

bioRxiv2020

Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales

A host of new technologies are under development to improve the quality and reproducibility of cryoEM grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms, (the fastest reported to date), to interrogate particle behaviour at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimise. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic silver bullet for improving the quality of every cryoEM sample preparation.

biochemistry↗

bioRxiv2019

A Cryo-EM Grid Preparation Device for Time-Resolved Structural Studies

Structural biology generally provides static snapshots of protein conformations that can inform on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualise, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. Recent advances in the resolution obtainable by electron microscopy (EM) and the broad range of samples that can be studied makes it ideally suited to time-resolved studies. Here we describe a cryo-electron microscopy grid preparation device that permits rapid mixing, voltage assisted spraying, and vitrification of samples. We show that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub 4 [A] 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 approaches.

biophysics↗