CLoNe: automated clustering based on local density neighborhoods for application to biomolecular structural ensembles.

MOTIVATION: Proteins are intrinsically dynamic entities. Flexibility sampling methods, such as molecular dynamics or those arising from integrative modeling strategies, are now commonplace and enable the study of molecular conformational landscapes in many contexts. Resulting structural ensembles increase in size as technological and algorithmic advancements take place, making their analysis increasingly demanding. In this regard, cluster analysis remains a go-to approach for their classification. However, many state-of-the-art algorithms are restricted to specific cluster properties. Combined with tedious parameter fine-tuning, cluster analysis of protein structural ensembles suffers from the lack of a generally applicable and easy to use clustering scheme. RESULTS: We present CLoNe, an original Python-based clustering scheme that builds on the Density Peaks algorithm of Rodriguez and Laio. CLoNe relies on a probabilistic analysis of local density distributions derived from nearest neighbors to find relevant clusters regardless of cluster shape, size, distribution and amount. We show its capabilities on many toy datasets with properties otherwise dividing state-of-the-art approaches and improves on the original algorithm in key aspects. Applied to structural ensembles, CLoNe was able to extract meaningful conformations from membrane binding events and ligand-binding pocket opening as well as identify dominant dimerization motifs or inter-domain organization. CLoNe additionally saves clusters as individual trajectories for further analysis and provides scripts for automated use with molecular visualization software. AVAILABILITY AND IMPLEMENTATION: www.epfl.ch/labs/lbm/resources, github.com/LBM-EPFL/CLoNe. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Modelling structures in cryo-EM maps.

Recent advances in structure determination of sub-cellular structures using cryo-electron microscopy and tomography have enabled us to understand their architecture in a more detailed manner and gain insight into their function. The choice of approach to use for atomic model building, fitting, refinement and validation in the 3D map resulting from these experiments depends primarily on the resolution of the map and the prior information on the corresponding model. Here, we survey some of such methods and approaches and highlight their uses in specific recent examples.

KAP1 is an antiparallel dimer with a functional asymmetry.

KAP1 (KRAB domain-associated protein 1) plays a fundamental role in regulating gene expression in mammalian cells by recruiting different transcription factors and altering the chromatin state. In doing so, KAP1 acts both as a platform for macromolecular interactions and as an E3 small ubiquitin modifier ligase. This work sheds light on the overall organization of the full-length protein combining solution scattering data, integrative modeling, and single-molecule experiments. We show that KAP1 is an elongated antiparallel dimer with an asymmetry at the C-terminal domains. This conformation is consistent with the finding that the Really Interesting New Gene (RING) domain contributes to KAP1 auto-SUMOylation. Importantly, this intrinsic asymmetry has key functional implications for the KAP1 network of interactions, as the heterochromatin protein 1 (HP1) occupies only one of the two putative HP1 binding sites on the KAP1 dimer, resulting in an unexpected stoichiometry, even in the context of chromatin fibers.

Disentangling constraints using viability evolution principles in integrative modeling of macromolecular assemblies.

Predicting the structure of large molecular assemblies remains a challenging task in structural biology when using integrative modeling approaches. One of the main issues stems from the treatment of heterogeneous experimental data used to predict the architecture of native complexes. We propose a new method, applied here for the first time to a set of symmetrical complexes, based on evolutionary computation that treats every available experimental input independently, bypassing the need to balance weight components assigned to aggregated fitness functions during optimization.

Clathrin regulates centrosome positioning by promoting acto-myosin cortical tension in C. elegans embryos.

Regulation of centrosome and spindle positioning is crucial for spatial cell division control. The one-cell Caenorhabditis elegans embryo has proven attractive for dissecting the mechanisms underlying centrosome and spindle positioning in a metazoan organism. Previous work revealed that these processes rely on an evolutionarily conserved force generator complex located at the cell cortex. This complex anchors the motor protein dynein, thus allowing cortical pulling forces to be exerted on astral microtubules emanating from microtubule organizing centers (MTOCs). Here, we report that the clathrin heavy chain CHC-1 negatively regulates pulling forces acting on centrosomes during interphase and on spindle poles during mitosis in one-cell C. elegans embryos. We establish a similar role for the cytokinesis/apoptosis/RNA-binding protein CAR-1 and uncover that CAR-1 is needed to maintain proper levels of CHC-1. We demonstrate that CHC-1 is necessary for normal organization of the cortical acto-myosin network and for full cortical tension. Furthermore, we establish that the centrosome positioning phenotype of embryos depleted of CHC-1 is alleviated by stabilizing the acto-myosin network. Conversely, we demonstrate that slight perturbations of the acto-myosin network in otherwise wild-type embryos results in excess centrosome movements resembling those in chc-1(RNAi) embryos. We developed a 2D computational model to simulate cortical rigidity-dependent pulling forces, which recapitulates the experimental data and further demonstrates that excess centrosome movements are produced at medium cortical rigidity values. Overall, our findings lead us to propose that clathrin plays a critical role in centrosome positioning by promoting acto-myosin cortical tension.