In several pathogenic bacteria, including Vibrio species, the filament of the bacterial flagellum is encased by a membranous sheath, an extension of the bacterial outer membrane. It has been proposed that having sheathed flagella permit bacteria to evade an immune response against flagellar components, suggesting a role in virulence. However, the molecular details of the interaction between sheath and filament, and how it impacts filament rotation, remain largely uncharacterized. Here, we combine single-particle cryo-electron microscopy, cryo-electron tomography, and genetic analyses to resolve the molecular architecture and biogenesis of the sheathed flagellum in Vibrio alginolyticus. We show that the flagellar filament forms a canonical 11-stranded supercoil made of the flagellin FlaD2 and enveloped by a bilayered sheath. We report that the filament surface is highly electronegative, suggesting that electrostatic repulsion between filament and sheath may reduce friction and supports high-speed flagellar rotation. We also show that the filament cap protein FliD possesses a unique domain in sheathed flagella, that may coordinate sheath assembly with filament elongation. Collectively, this structural insight into the structure of the Vibrio alginolyticus flagellum suggests a molecular mechanism for the rotation of sheathed flagella.

Abstract Drebrin modulates F-actin networks and links them to other intracellular components, regulating crucial processes including neuritogenesis, synaptic plasticity, virus internalisation and cancer invasion. Using single-particle cryo-EM we characterise drebrin’s interaction with F-actin through two separate conserved actin binding domains (ABD1 and ABD2), revealing structural bases for its F-actin-modulating properties. We describe a multimodal interaction where drebrin’s ABD1 can adopt two conformations and a long flexible loop connecting to ABD2 allows the two ABDs to occupy multiple relative positions along F-actin. The flexible loop connecting the two ABDs also confers some propensity to loosely bundle F-actin. Drebrin’s ABDs bind across multiple actin protomers and their subdomains and modify the longitudinal inter-protomer interface, explaining its F-actin stabilising properties. Furthermore, we show drebrin’s binding site on F-actin is shared with other critical actin-binding and regulatory proteins, explaining their competitive displacement.

Sorting nexin 9 (SNX9) is a membrane-binding scaffold protein that contributes to viral uptake and inflammation and is associated with worse outcomes in several cancers. It is involved in endocytosis of epidermal growth factor receptors, β1-integrin and membrane type 1 matrix metalloprotease, and formation of mitochondrial-derived vesicles. The SNX9 Bin-Amphiphysin-Rvs (BAR)-Phox homology (PX) domains bind phosphoinositide lipids and the Src homology 3 (SH3) domain interacts with dynamin and Neural-Wiskott Aldrich syndrome protein (N-WASP) to stimulate Arp2/3 complex-mediated actin polymerization. Here we use biolayer interferometry, cell-free reconstitution, and superresolution microscopy to analyze the specificity and activities of SNX9 at membranes. We find that more SNX9 can bind liposomes containing phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) and phosphatidylinositol (3)-phosphate (PI(3)P) compared with phosphatidylinositol (3,4)-bisphosphate (PI(3,4)P2), despite similar affinities. Actin assembly requires the network of both PX-BAR and SH3 interactions. Three-dimensional direct stochastic optical reconstruction microscopy on filopodia-like reconstitutions shows that SNX9 and related protein transducer of Cdc42-dependent actin assembly-1 (TOCA-1) can form both flat and ∼0.5 µm curved assemblies at actin incorporation sites. Finally, using cryo-electron tomography, we show that SNX9 builds both branched and bundled actin networks demonstrating its potential for multifunctional roles in actin remodeling.

Signaling between the endoplasmic reticulum (ER) and mitochondria regulates many of the seemingly disparate physiological functions that are damaged in neurodegenerative diseases such as Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). A number of studies have now demonstrated that ER-mitochondria signaling is perturbed in these diseases and there is evidence that this may be a driving mechanism in disease onset and progression. VAPB and PTPIP51 are ER-mitochondria tethering proteins; VAPB is an ER protein and PTPIP51 is an outer mitochondrial membrane protein and the two proteins interact to enable inter-organelle signaling. The VAPB-PTPIP51 interaction is disrupted in Alzheimer’s disease, Parkinson’s disease, FTD and ALS. Here we review the roles of VAPB and PTPIP51 in ER-mitochondria signaling and the mechanisms by which neurodegenerative disease insults may disrupt the VAPB-PTPIP51 interaction.

Kinesin-1 is a processive dimeric ATP-driven motor that transports vital intracellular cargos along microtubules (MTs). If not engaged in active transport, kinesin-1 limits futile ATP hydrolysis by adopting a compact autoinhibited conformation that involves an interaction between its C-terminal tail and the N-terminal motors domains. Here, using a chimeric kinesin-1 that fuses the N-terminal motor region to the tail and a tail variant unable to interact with the motors, we employed high-resolution cryo-EM in the presence of MTs to investigate elements of the mechanochemical cycle. We describe a missing structure for the proposed two-step allosteric mechanism of ADP release, the ATPase rate limiting step. It shows that MT association induces remodeling of the hydrogen bond network at the nucleotide binding site triggering removal of the Mg2+ ion from the Mg2+-ADP complex resulting in a strong MT-binding apo-like state before ADP dissociation. We further demonstrate that tail association does not directly affect this mechanism, nor the adoption of the ATP hydrolysis-competent conformation, nor neck linker docking/undocking, even when zippering the two motor domains. Based on this structural evidence, we propose a revised mechanism for tail-dependent kinesin-1 autoinhibition and suggest a possible explanation for its characteristic pausing behavior on MTs. ### Competing Interest Statement The authors have declared no competing interest.

Historically proteins that form highly polymeric and filamentous assemblies have been notoriously difficult to study using high resolution structural techniques. This has been due to several factors that include structural heterogeneity, their large molecular mass, and available yields. However, over the past decade we are now seeing a major shift towards atomic resolution insight and the study of more complex heterogenous samples and in situ/ex vivo examination of multi-subunit complexes. Although supported by developments in solid state nuclear magnetic resonance spectroscopy (ssNMR) and computational approaches, this has primarily been due to advances in cryogenic electron microscopy (cryo-EM). The study of eukaryotic microtubules and bacterial pili are good examples, and in this review, we will give an overview of the technical innovations that have enabled this transition and highlight the advancements that have been made for these two systems. Looking to the future we will also describe systems that remain difficult to study and where further technical breakthroughs are required.

Neurons navigate long distances and extend axons to form the complex circuitry of the mature brain. This depends on the coordinated response and continuous remodelling of the microtubule and F-actin networks in the axonal growth cone. Growth cone architecture remains poorly understood at nanoscales. We therefore investigated mouse hippocampal neuron growth cones using cryo-electron tomography to directly visualise their three-dimensional subcellular architecture with molecular detail. Our data show the hexagonal arrays of actin bundles that form filopodia penetrate the growth cone interior and terminate in the transition zone. We directly observe the modulation of these and other growth cone actin bundles by alteration of individual F-actin helical structures. Blunt-ended microtubules predominate in the growth cone, frequently contain lumenal particles and carry lattice defects. Investigation of the effect of absence of doublecortin, a neurodevelopmental cytoskeleton regulator, on growth cone cytoskeleton shows no major anomalies in overall growth cone organisation or in F-actin subpopulations. However, our data suggest that microtubules sustain more structural defects, highlighting the importance of microtubule integrity during growth cone migration. Summary statement Cryo-electron tomographic reconstruction of neuronal growth cone subdomains reveals distinctive F-actin and microtubule cytoskeleton architectures and modulation at molecular detail.

Plasmodium parasites cause malaria and are responsible annually for hundreds of thousands of deaths. Kinesins are a superfamily of microtubule-dependent ATPases that play important roles in the parasite replicative machinery, which is a potential target for antiparasite drugs. Kinesin-5, a molecular motor that cross-links microtubules, is an established antimitotic target in other disease contexts, but its mechanism in Plasmodium falciparum is unclear. Here, we characterized P. falciparum kinesin-5 (PfK5) using cryo-EM to determine the motor's nucleotide-dependent microtubule-bound structure and introduced 3D classification of individual motors into our microtubule image processing pipeline to maximize our structural insights. Despite sequence divergence in PfK5, the motor exhibits classical kinesin mechanochemistry, including ATP-induced subdomain rearrangement and cover neck bundle formation, consistent with its plus-ended directed motility. We also observed that an insertion in loop5 of the PfK5 motor domain creates a different environment in the well-characterized human kinesin-5 drug-binding site. Our data reveal the possibility for selective inhibition of PfK5 and can be used to inform future exploration of Plasmodium kinesins as antiparasite targets.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP-kinesin motor domain complex. KBP is a TPR-containing, crescent-shaped right-handed α-solenoid that sequesters the tubulin-binding surface of the kinesin motor domain, structurally distorting the motor domain and sterically blocking MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain and selective mutation of this extended binding surface disrupts KBP inhibition of kinesin transport in cells. The KBP-interacting surface of the motor domain contains motifs exclusively conserved in KBP-interacting kinesins, providing a basis for kinesin selectivity.### Competing Interest StatementThe authors have declared no competing interest.

Kinesin-binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg–Shprintzen syndrome. Cryo-electron microscopy (cryo-EM) has recently been used to reveal the structure of KBP alone (72 kDa) and in complex with the motor domain of the mitotic kinesin-12 KIF15 (110 kDa). KBP is an α-solenoid, tetratricopeptide-repeat protein that interacts with the microtubule-binding region of the kinesin motor domain and blocks microtubule attachment. Numerous challenges arose relating to the behavior of KBP and KBP–kinesin complexes during cryo-EM sample preparation. These included the partial denaturation of KBP by air–water interfaces, protein aggregation resulting from carbon interaction and preferential orientation. Sample preparation with a graphene oxide substrate enabled the eventual structure determination. Here, experiences with preparing these samples are detailed, bringing attention to some of the challenges and opportunities that are likely to arise from protein-surface interactions.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Microtubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo . Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Mi crotubule R ELION-based P ipeline (MiRP) . The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins. Abbreviations MiRP, Microtubule RELION-based Pipeline; cryo-EM, cryo-electron microscopy; MT, microtubule; CTF, contrast transfer function; PF, protofilament.

Subcellular compartmentalisation is necessary for eukaryotic cell function. Spatial and temporal regulation of kinesin activity is essential for building these local environments via control of intracellular cargo distribution. Kinesin-binding protein (KBP) interacts with a subset of kinesins via their motor domains, inhibits their microtubule (MT) attachment, and blocks their cellular function. However, its mechanisms of inhibition and selectivity have been unclear. Here we use cryo-electron microscopy to reveal the structure of KBP and of a KBP–kinesin motor domain complex. KBP is a tetratricopeptide repeat-containing, right-handed α-solenoid that sequesters the kinesin motor domain’s tubulin-binding surface, structurally distorting the motor domain and sterically blocking its MT attachment. KBP uses its α-solenoid concave face and edge loops to bind the kinesin motor domain, and selected structure-guided mutations disrupt KBP inhibition of kinesin transport in cells. The KBP-interacting motor domain surface contains motifs exclusively conserved in KBP-interacting kinesins, suggesting a basis for kinesin selectivity.

Department of Molecular and Cell Biology, University of Leicester, Lancaster Road, Leicester LE1 9HN, U.K. Institute of Structural and Molecular Biology, Birkbeck College, Malet Street, London WC1E 7HX, U.K. Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds LS2 9JT, U.K. Centre for Core Biotechnology Services, University of Leicester, University Road, Leicester, LE1 7RH, UK. Centre for Mechanochemical Cell Biology, Warwick Medical School, University of Warwick, Coventry CV4 7AL, U.K.

CAMSAP/Patronins regulate microtubule minus-end dynamics. Their end specificity is mediated by their CKK domains, which we proposed recognise specific tubulin conformations found at minus ends. To critically test this idea, we compared the human CAMSAP1 CKK domain (HsCKK) with a CKK domain from Naegleria gruberi (NgCKK), which lacks minus-end specificity. Here we report near-atomic cryo-electron microscopy structures of HsCKK- and NgCKK-microtubule complexes, which show that these CKK domains share the same protein fold, bind at the intradimer interprotofilament tubulin junction, but exhibit different footprints on microtubules. NMR experiments show that both HsCKK and NgCKK are remarkably rigid. However, whereas NgCKK binding does not alter the microtubule architecture, HsCKK remodels its microtubule interaction site and changes the underlying polymer structure because the tubulin lattice conformation is not optimal for its binding. Thus, in contrast to many MAPs, the HsCKK domain can differentiate subtly specific tubulin conformations to enable microtubule minus-end recognition.

NEK family kinases target EML4 to ensure faithful chromosome alignment during metaphase.

Microtubules are polar polymers, with minus and plus ends exhibiting differential dynamics and regulated by different cofactors. The evolutionarily conserved calmodulin-regulated spectrin-associated protein (CAMSAP) family are minus end binding proteins that modulate minus end microtubule dynamics. All CAMSAPs share a characteristic conserved CKK domain that defines microtubule minus end specificity. Previously we found that CKK binds microtubules at an intra-dimer inter-protofilament site and suggested a mechanism of specificity based on tubulin polymer conformation. To further investigate this proposed mechanism, we sought to structurally compare a microtubule minus end-preferring CKK with a CKK from the protist N.gruberi (NgCKK) that shows no end preference. Near-atomic resolution structures of 13 and 14 protofilament human CAMSAP1-CKK (HsCKK) and NgCKK decorated microtubules were determined using a new processing protocol and homogenous human embryonic kidney cell tubulin. NgCKK was found to have a similar fold and microtubule binding site to HsCKK, yet it showed minor loop modifications and subtly altered positioning relative to the microtubule lattice leading to a modified interface. Furthermore, unlike HsCKK, NgCKK was found to neither compress the microtubule lattice nor impose right-handed supertwist on microtubules. We suggest that these modified structural characteristics prevent NgCKK from sensing conformations of tubulin located uniquely at the microtubule minus end and, conversely, identify the features of HsCKK that confer its minus-end binding preference.

Background: Microtubules are dynamic protein filaments that are crucial for cell division and constitute key elements of the cytoskeleton.They are assembled from αβ-tubulin heterodimers that form hollow cylindrical structures.There is a large number of naturally occurring compounds that are known to interact with tubulin, including alkaloids, macrolides and peptides, which are collectively called microtubule-targeting agents (MTAs).Based on their activities, MTAs can be classified as microtubulestabilizing agents (MSAs) that enhance MT assembly, and microtubule-destabilizing agents (MDAs) that suppress MT assembly.The chemical structure of these drugs and their binding mode to microtubules varies greatly amongst each other and confer the ability to act either synergistically or competitively on microtubules.Owing to their effects on microtubule dynamics, MTAs are of great interest and widely used in a variety of medical applications as antiparasitic agents, herbicides and, most importantly, as chemotherapeutic drugs used for the treatment of cancer.In the past years, we and others solved the structures of a large number of different MTAs bound to tubulin to high resolution using X-ray crystallography.Very recently, however, with the advent of the "Resolution Revolution" in cryo-electron microscopy (cryo-EM), atomic structures of known MSAs bound to microtubules have also been obtained.These cryo-EM structures confirmed that the sites and modes of binding described in the previous X-ray crystallographic studies are similar in the context of the assembled microtubule but additionally explain the effects of MTAs on lattice parameters in microtubules and the lateral contacts between protofilaments, especially at the microtubule seam.Methods: Cryo-EM and model building. Results:We solved several high-resolution structures of microtubules bound to novel MSAs and identified differential binding modes in comparison to that revealed by X-ray crystallography.Conclusion: Cryo-EM and X-ray crystallography can be used in a complementary manner to investigate the molecular mechanism of action of MSAs in detail.

Microtubules are polar polymers, with minus and plus ends exhibiting differential dynamics and regulated by different cofactors. The evolutionarily conserved calmodulin-regulated spectrin-associated protein (CAMSAP) family are minus end binding proteins that modulate minus end microtubule dynamics. All CAMSAPs share a characteristic conserved CKK microtubule binding domain, which defines their minus end specificity. However the mechanism of this specificity is not understood. To shed light on this question, we determined the CKK structure by X-ray crystallography and characterised CAMSAP1 and 3's CKK binding site on microtubules by cryo-EM. The CAMSAP CKK binds at a previously undescribed binding site at the microtubule intra-dimer inter-protofilament interface. TIRF microscopy was used to confirm the contributions of conserved residues at the CKK-microtubule interface. The nucleotide state of tubulin was also shown not to influence minus-end specificity. Interestingly, CKK binding imposes a right-handed 'supertwist', on microtubule protofilaments, even on the taxol-stabilized microtubules used for our reconstructions. Further cryo-EM investigations, including of a CKK mutant, and another CKK domain with reduced minus-end specificity, supports the possibility that subtle alterations in CKK positioning relative to tubulin polymer confer minus-end specificity. Cryo-electron tomography of minus-ends reveals curved lattice to sheet regions that retain lateral protofilament interactions and present unique minus-end tubulin conformations to which CAMSAPs may preferentially bind.

EML4 is a microtubule-associated protein that promotes microtubule stability. We show here that EML4 is distributed as punctate foci along the microtubule lattice in interphase but exhibits reduced association with spindle microtubules in mitosis. Microtubule sedimentation and cryo-electron microscopy and 3D reconstruction reveal that EML4 binds via its basic N-terminal domain to the acidic C-terminal tails of α- and β-tubulin on the microtubule surface. The mitotic kinases Nek6 and Nek7 can phosphorylate EML4 N-terminal domain at S144 and S146 in vitro, and depletion of these kinases leads to increased EML4 binding to microtubules in mitosis. An S144A-S146A double mutant not only binds inappropriately to mitotic microtubules but also interferes with chromosome congression. Meanwhile, constitutive activation of Nek6 or Nek7 reduces EML4 association with interphase microtubules. Together, these data support a model in which Nek6 and Nek7-dependent phosphorylation promotes dissociation of EML4 from microtubules in mitosis thereby altering microtubule dynamics to enable chromosome congression.