Showing papers by "José María Carazo published in 2018"

Automatic local resolution-based sharpening of cryo-EM maps.

Abstract: Recent technological advances and computational developments, have allowed the reconstruction of cryo-EM maps at near-atomic resolution structures. Cryo-EM maps benefit significantly of a 9postprocessing9 step, normally referred to as 9sharpening9, that tends to increase signal at medium/high resolution. Here, we propose a new method for local sharpening of volumes generated by cryo-EM. The algorithm (LocalDeblur) is based on a local resolution-guided Wiener restoration approach, does not need any prior atomic model and it avoids artificial structure factor corrections. LocalDeblur is fully automatic and parameter free. We show that the new method significantly and quantitatively improving map quality and interpretability, especially in cases of broad local resolution changes (as is often the case of membrane proteins).

Dynamic kinetochore size regulation promotes microtubule capture and chromosome biorientation in mitosis.

Abstract: Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. The outer layer of kinetochores transiently expands in early mitosis to form a fibrous corona, and compacts following microtubule capture. Here we show that the dynein adaptor Spindly and the RZZ (ROD-Zwilch-ZW10) complex drive kinetochore expansion in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ-Spindly complexes into a filamentous meshwork in cells and in vitro. Concurrent with kinetochore expansion, Spindly potentiates kinetochore compaction by recruiting dynein via three conserved short linear motifs. Expanded kinetochores unable to compact engage in extensive, long-lived lateral microtubule interactions that persist to metaphase, and result in merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.

Dynamic Kinetochore Size Regulation Promotes Microtubule Capture and Chromosome Biorientation in Mitosis

Abstract: Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. An outer layer of the kinetochore known as the fibrous corona transiently expands in early mitosis and disassembles upon microtubule capture. Neither the functional importance nor the mechanistic basis for this are known. Here we show that the dynein adaptor Spindly and the RZZ kinetochore complex drive fibrous corona formation in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ:Spindly complexes into a corona-like meshwork in cells and in vitro. Concurrent with corona expansion, Spindly potentiates corona shedding by recruiting dynein via three conserved short linear motifs. Expanded, non-sheddable fibrous coronas engage in extensive, long-lived lateral microtubule interactions that persist to metaphase and result in fused sister kinetochores, formation of merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.

X-ray structure of full-length human RuvB-Like 2 - mechanistic insights into coupling between ATP binding and mechanical action

Abstract: RuvB-Like transcription factors function in cell cycle regulation, development and human disease, such as cancer and heart hyperplasia The mechanisms that regulate adenosine triphosphate (ATP)-dependent activity, oligomerization and post-translational modifications in this family of enzymes are yet unknown We present the first crystallographic structure of full-length human RuvBL2 which provides novel insights into its mechanistic action and biology The ring-shaped hexameric RuvBL2 structure presented here resolves for the first time the mobile domain II of the human protein, which is responsible for protein-protein interactions and ATPase activity regulation Structural analysis suggests how ATP binding may lead to domain II motion through interactions with conserved N-terminal loop histidine residues Furthermore, a comparison between hsRuvBL1 and 2 shows differences in surface charge distribution that may account for previously described differences in regulation Analytical ultracentrifugation and cryo electron microscopy analyses performed on hsRuvBL2 highlight an oligomer plasticity that possibly reflects different physiological conformations of the protein in the cell, as well as that single-stranded DNA (ssDNA) can promote the oligomerization of monomeric hsRuvBL2 Based on these findings, we propose a mechanism for ATP binding and domain II conformational change coupling

Scipion web tools: Easy to use cryo-EM image processing over the web.

Abstract: Macromolecular structural determination by Electron Microscopy under cryogenic conditions is revolutionizing the field of structural biology, interesting a large community of potential users. Still, the path from raw images to density maps is complex, and sophisticated image processing suites are required in this process, often demanding the installation and understanding of different software packages. Here, we present Scipion Web Tools, a web-based set of tools/workflows derived from the Scipion image processing framework, specially tailored to nonexpert users in need of very precise answers at several key stages of the structural elucidation process.

Using Scipion for stream image processing at cryo-EM facilities

Abstract: (a) Escuela Politécnica Superior, Universidad Autónoma de Madrid, 28049 Cantoblanco, Madrid, Spain. (b) Biocomputing Unit, National Center for Biotechnology (CSIC), C/ Darwin, 3, Campus Universidad Autónoma, 28049 Cantoblanco, Madrid, Spain. (c) Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden (e) Diamond Light Source, Harwell Science and Innovation Campus, Didcot OX11 0DE, England

ENRICH: a fast method to improve the quality of flexible macromolecular reconstructions

Abstract: Cryo-electron microscopy using single particle analysis requires the computational averaging of thousands of projection images captured from identical macromolecules. However, macromolecules usually present some degree of flexibility showing different conformations. Computational approaches are then required to classify heterogeneous single particle images into homogeneous sets corresponding to different structural states. Nonetheless, sometimes the attainable resolution of reconstructions obtained from these smaller homogeneous sets is compromised because of reduced number of particles or lack of images at certain macromolecular orientations. In these situations, the current solution to improve map resolution is returning to the electron microscope and collect more data. In this work, we present a fast approach to partially overcome this limitation for heterogeneous data sets. Our method is based on deforming and then moving particles between different conformations using an optical flow approach. Particles are then merged into a unique conformation obtaining reconstructions with improved resolution, contrast and signal-to-noise ratio, then, partially circumventing many issues that impact obtaining high quality reconstructions from small data sets. We present experimental results that show clear improvements in the quality of obtained 3D maps, however, there are also limits to this approach, which we discuss in the manuscript.