Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions

Kinesin-binding protein (KBP) is an important selective inhibitor of specific kinesin family members and its genetic disruption causes Goldberg-Shprintzen syndrome (GOSHS). We recently used cryo-electron microscopy 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 behaviour of KBP and KBP-kinesin complexes during cryo-EM sample preparation. This included 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 eventual structure determination. Here we detail our experiences with preparing these samples, bringing attention to some of the challenges and opportunities likely arising from protein-surface interactions

A microtubule RELION-based pipeline for cryo-EM image processing

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, Microtubule RELION-based Pipeline (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

Visualising the cytoskeletal machinery in neuronal growth cones using cryo-electron tomography

Neurons 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 threedimensional subcellular architecture with molecular detail. Our data show the hexagonal arrays of actin bundles that form filopodia penetrate and terminate deep in the growth cone interior. We directly observe the modulation of these and other growth cone actin bundles by alteration of individual F-actin helical structures. Microtubules with blunt, slightly flared, or gently curved ends 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

Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition

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 anti-parasite drugs. Kinesin-5, a molecular motor that crosslinks microtubules, is an established anti-mitotic targets in other disease contexts, but its mechanism in P. falciparum is unclear. Here, we characterised 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 maximise 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 characterised 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 anti-parasite targets

The mechanism of kinesin inhibition by kinesin binding protein

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, 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

Improving health outcome for young people with long term conditions: The role of digital communication in current and future patient-clinician communication for NHS providers of specialist clinical services for young people receiving specialist clinical services:LYNC study protocol.

Background: Young people living with long term conditions are vulnerable to health service disengagement. This endangers their long term health. Studies report requests for digital forms of communication - email, text, social media - with their health care team. Digital clinical communication is troublesome for the UK NHS. Aim: To present the research protocol for evaluating the impacts and outcomes of digital clinical communications for young people living with long term conditions and provide critical analysis of their use, monitoring and evaluation by NHS providers. Methods: The research involves: 1) Patient and Public Involvement activities with 16-24 year olds with and without long term health conditions; 2) six literature reviews; 3) case studies – the main empirical part of the study – and 4) synthesis and a consensus meeting. Case studies use a mixed methods design. Interviews and non-participant observation of practitioners and patients communicating in up to 20 specialist clinical settings will be combined with data, aggregated at the case level (non-identifiable patient data), on a range of clinical outcomes meaningful within the case and across cases. We will describe the use of digital clinical communication from the perspective of patients, clinical staff, support staff and managers, interviewing up to 15 young people and 15 staff per case study. Outcome data includes emergency admissions, A&E attendance and DNA rates. Case studies will be analysed to understand impacts of digital clinical communication on patient health outcomes, health care costs and consumption, ethics and patient safety

Reporting and interpreting effect size in quantitative agricultural education research

The Journal of Agricultural Education (JAE) requires authors to follow the guidelines stated in the Publication Manual of the American Psychological Association [APA] (2009) in preparing research manuscripts, and to utilize accepted research and statistical methods in conducting quantitative research studies. The APA recommends the reporting of effect sizes in quantitative research, when appropriate. JAE now requires the reporting of effect size when reporting statistical significance in quantitative manuscripts. The purposes of this manuscript are to describe the research foundation supporting the reporting of effect size in quantitative research and to provide examples of how to calculate effect size for some of the most common statistical analyses utilized in agricultural education research. Recommendations for appropriate effect size measures and interpretation are included. The assumptions and limitations inherent in the reporting of effect size in research are also incorporated