A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (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 behavior 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 optimize. 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.

Rapid advances in experimental biophysical techniques are generating a wealth of information about the mechanical operation of the cellular cytoskeleton and its motors. However, each of these tools typically provides only a limited piece of a highly complex puzzle. There is a need to develop new computational tools that can integrate these data together into a central model. Here we discuss the experimental advances alongside the computational tools, and propose how these could be developed to successfully combine the emerging structural and dynamic experimental data on cytoskeletal motors. We consider examples of both single motors and arrays of motors within a biological cell.This article is categorized under: Structure and Mechanism > Molecular Structures Data Science > Computer Algorithms and Programming Data Science > Visualization

Myosin 5a transports cellular cargos along actin filaments towards the cell periphery. Its long lever plays a key role in determining the large size of its powerstoke, stepping distance along F-actin, ability to bear load and its regulation by Ca ²⁺ . Despite this, little is known about the physical properties of the lever and how they contribute to the mechanics of walking. Using a combination of cryo-electron microscopy and molecular dynamics simulations, we resolved the first structure of myosin 5a comprising the motor domain and full-length lever (subfragment-1) bound to actin. From the flexibility captured in the cryo-electron microscopy data, we were able to characterise the stiffness of the lever. Here, we demonstrate how the structure and flexibility of the lever contribute to the regulation and walking behaviour of myosin 5a.

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.

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.

The manchette is a transient microtubule (MT)-based structure that is vital for the correct shaping of sperm during spermiogenesis. Throughout spermiogenesis, the manchette retains structural integrity for several days, raising the question of how its MTs are regulated. Here, using cryo-electron tomography of manchettes isolated from rat testes, we find that manchette MT ends are structurally diverse. We show that the MT-binding protein CLASP2 is present throughout the manchette and likely regulates both MT ends. Using cryo-electron microscopy single particle analysis and super-resolution microscopy, we reveal that SPACA9 and MNMIP1 (SH3D21) bind to the seam of manchette MTs from the luminal side. SPACA9 binds to both α- and β-tubulin of protofilament 1 but does not interact directly with protofilament 13, while MNMIP1 binds directly to protofilament 13. MNMIP1 further extends and threads through the MT lattice at the seam. Our study reveals a novel seam MT inner protein complex with a unique binding mode, providing a plausible explanation for MT regulation that maintains manchette integrity over an extended period.