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1.
Elife ; 122024 Apr 10.
Article in English | MEDLINE | ID: mdl-38597186

ABSTRACT

Epithelial intercellular adhesion molecule (ICAM)-1 is apically polarized, interacts with, and guides leukocytes across epithelial barriers. Polarized hepatic epithelia organize their apical membrane domain into bile canaliculi and ducts, which are not accessible to circulating immune cells but that nevertheless confine most of ICAM-1. Here, by analyzing ICAM-1_KO human hepatic cells, liver organoids from ICAM-1_KO mice and rescue-of-function experiments, we show that ICAM-1 regulates epithelial apicobasal polarity in a leukocyte adhesion-independent manner. ICAM-1 signals to an actomyosin network at the base of canalicular microvilli, thereby controlling the dynamics and size of bile canalicular-like structures. We identified the scaffolding protein EBP50/NHERF1/SLC9A3R1, which connects membrane proteins with the underlying actin cytoskeleton, in the proximity interactome of ICAM-1. EBP50 and ICAM-1 form nano-scale domains that overlap in microvilli, from which ICAM-1 regulates EBP50 nano-organization. Indeed, EBP50 expression is required for ICAM-1-mediated control of BC morphogenesis and actomyosin. Our findings indicate that ICAM-1 regulates the dynamics of epithelial apical membrane domains beyond its role as a heterotypic cell-cell adhesion molecule and reveal potential therapeutic strategies for preserving epithelial architecture during inflammatory stress.


Subject(s)
Actomyosin , Intercellular Adhesion Molecule-1 , Animals , Mice , Humans , Actomyosin/metabolism , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Epithelial Cells/metabolism , Hepatocytes/metabolism , Liver/metabolism , Actin Cytoskeleton/metabolism , Leukocytes/metabolism , Cell Polarity
2.
Micromachines (Basel) ; 14(10)2023 Sep 26.
Article in English | MEDLINE | ID: mdl-37893272

ABSTRACT

Cryogenic electron microscopy (Cryo-EM) has been established as one of the key players in structural biology. It can reconstruct a 3D model of a sample at a near-atomic resolution. With the increasing number of facilities, faster microscopes, and new imaging techniques, there is a growing demand for algorithms and programs able to process the so-called movie data produced by the microscopes in real time while preserving a high resolution and maximal information. In this article, we conduct a comparative analysis of the quality and performance of the most commonly used software for movie alignment. More precisely, we compare the most recent versions of FlexAlign (Xmipp v3.23.03), MotionCor2 (v1.6.4), Relion MotionCor (v4.0-beta), Warp (v1.0.9), and CryoSPARC (v4.0.3). We tested the quality of the alignment using generated phantom data, as well as real datasets, comparing the alignment precision, power spectra density, and performance scaling of each program.

3.
Int J Mol Sci ; 24(18)2023 Sep 18.
Article in English | MEDLINE | ID: mdl-37762547

ABSTRACT

Macromolecular assemblies, such as protein complexes, undergo continuous structural dynamics, including global reconfigurations critical for their function. Two fast analytical methods are widely used to study these global dynamics, namely elastic network model normal mode analysis and principal component analysis of ensembles of structures. These approaches have found wide use in various computational studies, driving the development of complex pipelines in several software packages. One common theme has been conformational sampling through hybrid simulations incorporating all-atom molecular dynamics and global modes of motion. However, wide functionality is only available for experienced programmers with limited capabilities for other users. We have, therefore, integrated one popular and extensively developed software for such analyses, the ProDy Python application programming interface, into the Scipion workflow engine. This enables a wider range of users to access a complete range of macromolecular dynamics pipelines beyond the core functionalities available in its command-line applications and the normal mode wizard in VMD. The new protocols and pipelines can be further expanded and integrated into larger workflows, together with other software packages for cryo-electron microscopy image analysis and molecular simulations. We present the resulting plugin, Scipion-EM-ProDy, in detail, highlighting the rich functionality made available by its development.


Subject(s)
Image Processing, Computer-Assisted , Cryoelectron Microscopy , Workflow , Databases, Factual , Motion
4.
mBio ; 14(2): e0002323, 2023 04 25.
Article in English | MEDLINE | ID: mdl-36786587

ABSTRACT

Fijiviruses replicate and package their genomes within viroplasms in a process involving RNA-RNA and RNA-protein interactions. Here, we demonstrate that the 24 C-terminal residues (C-arm) of the P9-1 major viroplasm protein of the mal de Río Cuarto virus (MRCV) are required for its multimerization and the formation of viroplasm-like structures. Using an integrative structural approach, the C-arm was found to be dispensable for P9-1 dimer assembly but essential for the formation of pentamers and hexamers of dimers (decamers and dodecamers), which favored RNA binding. Although both P9-1 and P9-1ΔC-arm catalyzed ATP with similar activities, an RNA-stimulated ATPase activity was only detected in the full-length protein, indicating a C-arm-mediated interaction between the ATP catalytic site and the allosteric RNA binding sites in the (do)decameric assemblies. A stronger preference to bind phosphate moieties in the decamer was predicted, suggesting that the allosteric modulation of ATPase activity by RNA is favored in this structural conformation. Our work reveals the structural versatility of a fijivirus major viroplasm protein and provides clues to its mechanism of action. IMPORTANCE The mal de Río Cuarto virus (MRCV) causes an important maize disease in Argentina. MRCV replicates in several species of Gramineae plants and planthopper vectors. The viral factories, also called viroplasms, have been studied in detail in animal reovirids. This work reveals that a major viroplasm protein of MRCV forms previously unidentified structural arrangements and provides evidence that it may simultaneously adopt two distinct quaternary assemblies. Furthermore, our work uncovers an allosteric communication between the ATP and RNA binding sites that is favored in the multimeric arrangements. Our results contribute to the understanding of plant reovirids viroplasm structure and function and pave the way for the design of antiviral strategies for disease control.


Subject(s)
Reoviridae , Viral Replication Compartments , Animals , RNA/metabolism , Reoviridae/chemistry , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism
5.
Biol Imaging ; 3: e13, 2023.
Article in English | MEDLINE | ID: mdl-38510163

ABSTRACT

Image-processing pipelines require the design of complex workflows combining many different steps that bring the raw acquired data to a final result with biological meaning. In the image-processing domain of cryo-electron microscopy single-particle analysis (cryo-EM SPA), hundreds of steps must be performed to obtain the three-dimensional structure of a biological macromolecule by integrating data spread over thousands of micrographs containing millions of copies of allegedly the same macromolecule. The execution of such complicated workflows demands a specific tool to keep track of all these steps performed. Additionally, due to the extremely low signal-to-noise ratio (SNR), the estimation of any image parameter is heavily affected by noise resulting in a significant fraction of incorrect estimates. Although low SNR and processing millions of images by hundreds of sequential steps requiring substantial computational resources are specific to cryo-EM, these characteristics may be shared by other biological imaging domains. Here, we present Scipion, a Python generic open-source workflow engine specifically adapted for image processing. Its main characteristics are: (a) interoperability, (b) smart object model, (c) gluing operations, (d) comparison operations, (e) wide set of domain-specific operations, (f) execution in streaming, (g) smooth integration in high-performance computing environments, (h) execution with and without graphical capabilities, (i) flexible visualization, (j) user authentication and private access to private data, (k) scripting capabilities, (l) high performance, (m) traceability, (n) reproducibility, (o) self-reporting, (p) reusability, (q) extensibility, (r) software updates, and (s) non-restrictive software licensing.

7.
Chem Rev ; 122(17): 13915-13951, 2022 09 14.
Article in English | MEDLINE | ID: mdl-35785962

ABSTRACT

Cryo-electron microscopy (CryoEM) has become a vital technique in structural biology. It is an interdisciplinary field that takes advantage of advances in biochemistry, physics, and image processing, among other disciplines. Innovations in these three basic pillars have contributed to the boosting of CryoEM in the past decade. This work reviews the main contributions in image processing to the current reconstruction workflow of single particle analysis (SPA) by CryoEM. Our review emphasizes the time evolution of the algorithms across the different steps of the workflow differentiating between two groups of approaches: analytical methods and deep learning algorithms. We present an analysis of the current state of the art. Finally, we discuss the emerging problems and challenges still to be addressed in the evolution of CryoEM image processing methods in SPA.


Subject(s)
Image Processing, Computer-Assisted , Single Molecule Imaging , Algorithms , Cryoelectron Microscopy/methods , Image Processing, Computer-Assisted/methods
8.
PLoS Pathog ; 18(7): e1010631, 2022 07.
Article in English | MEDLINE | ID: mdl-35816514

ABSTRACT

The S:A222V point mutation, within the G clade, was characteristic of the 20E (EU1) SARS-CoV-2 variant identified in Spain in early summer 2020. This mutation has since reappeared in the Delta subvariant AY.4.2, raising questions about its specific effect on viral infection. We report combined serological, functional, structural and computational studies characterizing the impact of this mutation. Our results reveal that S:A222V promotes an increased RBD opening and slightly increases ACE2 binding as compared to the parent S:D614G clade. Finally, S:A222V does not reduce sera neutralization capacity, suggesting it does not affect vaccine effectiveness.


Subject(s)
COVID-19 , SARS-CoV-2 , Angiotensin-Converting Enzyme 2/genetics , COVID-19/genetics , Genetic Background , Humans , Mutation , Peptidyl-Dipeptidase A/metabolism , Protein Binding , Receptors, Virus/metabolism , SARS-CoV-2/genetics , Spike Glycoprotein, Coronavirus/metabolism
9.
Acta Crystallogr D Struct Biol ; 78(Pt 4): 399-409, 2022 Apr 01.
Article in English | MEDLINE | ID: mdl-35362464

ABSTRACT

Cryo-electron microscopy (cryoEM) has become a well established technique with the potential to produce structures of large and dynamic supramolecular complexes that are not amenable to traditional approaches for studying structure and dynamics. The size and low resolution of such molecular systems often make structural modelling and molecular dynamics simulations challenging and computationally expensive. This, together with the growing wealth of structural data arising from cryoEM and other structural biology methods, has driven a trend in the computational biophysics community towards the development of new pipelines for analysing global dynamics using coarse-grained models and methods. At the centre of this trend has been a return to elastic network models, normal mode analysis (NMA) and ensemble analyses such as principal component analysis, and the growth of hybrid simulation methodologies that make use of them. Here, this field is reviewed with a focus on ProDy, the Python application programming interface for protein dynamics, which has been developed over the last decade. Two key developments in this area are highlighted: (i) ensemble NMA towards extracting and comparing the signature dynamics of homologous structures, aided by the recent SignDy pipeline, and (ii) pseudoatom fitting for more efficient global dynamics analyses of large and low-resolution supramolecular assemblies from cryoEM, revisited in the CryoDy pipeline. It is believed that such a renewal and extension of old models and methods in new pipelines will be critical for driving the field forward into the next cryoEM revolution.


Subject(s)
Molecular Dynamics Simulation , Cryoelectron Microscopy/methods , Principal Component Analysis
10.
IUCrJ ; 8(Pt 6): 992-1005, 2021 Nov 01.
Article in English | MEDLINE | ID: mdl-34804551

ABSTRACT

Structural biology has evolved greatly due to the advances introduced in fields like electron microscopy. This image-capturing technique, combined with improved algorithms and current data processing software, allows the recovery of different conformational states of a macromolecule, opening new possibilities for the study of its flexibility and dynamic events. However, the ensemble analysis of these different conformations, and in particular their placement into a common variable space in which the differences and similarities can be easily recognized, is not an easy matter. To simplify the analysis of continuous heterogeneity data, this work proposes a new automatic algorithm that relies on a mathematical basis defined over the sphere to estimate the deformation fields describing conformational transitions among different structures. Thanks to the approximation of these deformation fields, it is possible to describe the forces acting on the molecules due to the presence of different motions. It is also possible to represent and compare several structures in a low-dimensional mapping, which summarizes the structural characteristics of different states. All these analyses are integrated into a common framework, providing the user with the ability to combine them seamlessly. In addition, this new approach is a significant step forward compared with principal component analysis and normal mode analysis of cryo-electron microscopy maps, avoiding the need to select components or modes and producing localized analysis.

11.
Proc Natl Acad Sci U S A ; 118(37)2021 09 14.
Article in English | MEDLINE | ID: mdl-34504018

ABSTRACT

During activation the platelet cytoskeleton is reorganized, inducing adhesion to the extracellular matrix and cell spreading. These processes are critical for wound healing and clot formation. Initially, this task relies on the formation of strong cellular-extracellular matrix interactions, exposed in subendothelial lesions. Despite the medical relevance of these processes, there is a lack of high-resolution structural information on the platelet cytoskeleton controlling cell spreading and adhesion. Here, we present in situ structural analysis of membrane receptors and the underlying cytoskeleton in platelet protrusions by applying cryoelectron tomography to intact platelets. We utilized three-dimensional averaging procedures to study receptors at the plasma membrane. Analysis of substrate interaction-free receptors yielded one main structural class resolved to 26 Å, resembling the αIIbß3 integrin folded conformation. Furthermore, structural analysis of the actin network in pseudopodia indicates a nonuniform polarity of filaments. This organization would allow generation of the contractile forces required for integrin-mediated cell adhesion.


Subject(s)
Actin Cytoskeleton , Actins/chemistry , Blood Platelets/physiology , Cell Membrane/metabolism , Cell Surface Extensions/physiology , Platelet Glycoprotein GPIIb-IIIa Complex/chemistry , Actins/metabolism , Cell Adhesion , Humans , Platelet Activation , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism
12.
Commun Biol ; 4(1): 874, 2021 07 15.
Article in English | MEDLINE | ID: mdl-34267316

ABSTRACT

Cryo-EM maps are valuable sources of information for protein structure modeling. However, due to the loss of contrast at high frequencies, they generally need to be post-processed to improve their interpretability. Most popular approaches, based on global B-factor correction, suffer from limitations. For instance, they ignore the heterogeneity in the map local quality that reconstructions tend to exhibit. Aiming to overcome these problems, we present DeepEMhancer, a deep learning approach designed to perform automatic post-processing of cryo-EM maps. Trained on a dataset of pairs of experimental maps and maps sharpened using their respective atomic models, DeepEMhancer has learned how to post-process experimental maps performing masking-like and sharpening-like operations in a single step. DeepEMhancer was evaluated on a testing set of 20 different experimental maps, showing its ability to reduce noise levels and obtain more detailed versions of the experimental maps. Additionally, we illustrated the benefits of DeepEMhancer on the structure of the SARS-CoV-2 RNA polymerase.


Subject(s)
Cryoelectron Microscopy/instrumentation , DNA-Directed RNA Polymerases/ultrastructure , Deep Learning , SARS-CoV-2/ultrastructure , Viral Proteins/ultrastructure
13.
J Struct Biol ; 213(3): 107771, 2021 09.
Article in English | MEDLINE | ID: mdl-34324977

ABSTRACT

The quality of a 3D map produced by the single-particle analysis method is highly dependent on an accurate assignment of orientations to the many experimental images. However, the problem's complexity implies the presence of several local minima in the optimized goal functions. Consequently, validation methods to confirm the angular assignment are very useful to yield higher-resolution 3D maps. In this work, we present a graph-signal-processing-based methodology that analyzes the correlation landscape as a function of the orientation, an approach allowing the estimation of the assigned orientations' reliability. Using this method, we may identify low-reliability images that probably incorrectly contribute to the final 3D reconstruction.


Subject(s)
Single Molecule Imaging , Cryoelectron Microscopy/methods , Reproducibility of Results
14.
Acta Crystallogr D Struct Biol ; 77(Pt 6): 835-839, 2021 Jun 01.
Article in English | MEDLINE | ID: mdl-34076596

ABSTRACT

Principal component analysis (PCA) has been widely proposed to analyze flexibility and heterogeneity in cryo-electron microscopy (cryoEM). In this paper, it is argued that (i) PCA is an excellent technique to describe continuous flexibility at low resolution (but not so much at high resolution) and (ii) PCA components should be analyzed in a concerted manner (and not independently).


Subject(s)
Cryoelectron Microscopy/methods , Image Processing, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Macromolecular Substances/chemistry , Models, Molecular , Principal Component Analysis/methods
15.
Methods Mol Biol ; 2305: 257-289, 2021.
Article in English | MEDLINE | ID: mdl-33950394

ABSTRACT

Cryo-electron microscopy has established as a mature structural biology technique to elucidate the three-dimensional structure of biological macromolecules. The Coulomb potential of the sample is imaged by an electron beam, and fast semi-conductor detectors produce movies of the sample under study. These movies have to be further processed by a whole pipeline of image-processing algorithms that produce the final structure of the macromolecule. In this chapter, we illustrate this whole processing pipeline putting in value the strength of "meta algorithms," which are the combination of several algorithms, each one with different mathematical rationale, in order to distinguish correctly from incorrectly estimated parameters. We show how this strategy leads to superior performance of the whole pipeline as well as more confident assessments about the reconstructed structures. The "meta algorithms" strategy is common to many fields and, in particular, it has provided excellent results in bioinformatics. We illustrate this combination using the workflow engine, Scipion.


Subject(s)
Algorithms , Cryoelectron Microscopy/methods , Image Processing, Computer-Assisted/methods , Imaging, Three-Dimensional/methods , Single Molecule Imaging/methods , Computational Biology , Macromolecular Substances/ultrastructure , Molecular Biology/methods , Workflow
16.
Bioinformatics ; 37(22): 4258-4260, 2021 11 18.
Article in English | MEDLINE | ID: mdl-34014278

ABSTRACT

SUMMARY: The web platform 3DBionotes-WS integrates multiple web services and an interactive web viewer to provide a unified environment in which biological annotations can be analyzed in their structural context. Since the COVID-19 outbreak, new structural data from many viral proteins have been provided at a very fast pace. This effort includes many cryogenic electron microscopy (cryo-EM) studies, together with more traditional ones (X-rays, NMR), using several modeling approaches and complemented with structural predictions. At the same time, a plethora of new genomics and interactomics information (including fragment screening and structure-based virtual screening efforts) have been made available from different servers. In this context, we have developed 3DBionotes-COVID-19 as an answer to: (i) the need to explore multiomics data in a unified context with a special focus on structural information and (ii) the drive to incorporate quality measurements, especially in the form of advanced validation metrics for cryo-EM. AVAILABILITY AND IMPLEMENTATION: https://3dbionotes.cnb.csic.es/ws/covid19. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Subject(s)
COVID-19 , Software , Humans , Genomics
18.
Nat Commun ; 12(1): 939, 2021 02 11.
Article in English | MEDLINE | ID: mdl-33574245

ABSTRACT

Coiled-coil protein origami (CCPO) is a modular strategy for the de novo design of polypeptide nanostructures. CCPO folds are defined by the sequential order of concatenated orthogonal coiled-coil (CC) dimer-forming peptides, where a single-chain protein is programmed to fold into a polyhedral cage. Self-assembly of CC-based nanostructures from several chains, similarly as in DNA nanotechnology, could facilitate the design of more complex assemblies and the introduction of functionalities. Here, we show the design of a de novo triangular bipyramid fold comprising 18 CC-forming segments and define the strategy for the two-chain self-assembly of the bipyramidal cage from asymmetric and pseudo-symmetric pre-organised structural modules. In addition, by introducing a protease cleavage site and masking the interfacial CC-forming segments in the two-chain bipyramidal cage, we devise a proteolysis-mediated conformational switch. This strategy could be extended to other modular protein folds, facilitating the construction of dynamic multi-chain CC-based complexes.


Subject(s)
Protein Domains , Protein Folding , Protein Multimerization , Proteins/chemistry , DNA/chemistry , Models, Molecular , Nanostructures/chemistry , Nanotechnology , Peptides/chemistry , Protein Conformation , Protein Engineering , Proteins/genetics
19.
Nat Commun ; 12(1): 42, 2021 01 04.
Article in English | MEDLINE | ID: mdl-33397925

ABSTRACT

In recent years, advances in cryoEM have dramatically increased the resolution of reconstructions and, with it, the number of solved atomic models. It is widely accepted that the quality of cryoEM maps varies locally; therefore, the evaluation of the maps-derived structural models must be done locally as well. In this article, a method for the local analysis of the map-to-model fit is presented. The algorithm uses a comparison of two local resolution maps. The first is the local FSC (Fourier shell correlation) between the full map and the model, while the second is calculated between the half maps normally used in typical single particle analysis workflows. We call the quality measure "FSC-Q", and it is a quantitative estimation of how much of the model is supported by the signal content of the map. Furthermore, we show that FSC-Q may be helpful to detect overfitting. It can be used to complement other methods, such as the Q-score method that estimates the resolvability of atoms.


Subject(s)
Algorithms , Cryoelectron Microscopy , Fourier Analysis , Models, Molecular , Receptors, G-Protein-Coupled/chemistry , Spike Glycoprotein, Coronavirus/chemistry
20.
Prog Biophys Mol Biol ; 160: 43-52, 2021 03.
Article in English | MEDLINE | ID: mdl-32470354

ABSTRACT

Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex. However, data processing that applies symmetry can average out asymmetric features and consequently data analysis methods are required to resolve asymmetric structural features. Scipion is a cryo-EM image processing framework that integrates functions from different image processing packages as plugins. To extend its functionality for handling symmetry mismatches, we present here a Scipion plugin termed LocalRec implementing the localized reconstruction method. When tested on an adenovirus data set, the plugin enables resolving the symmetry-mismatched trimeric fibre bound to the five-fold vertices of the capsid. Furthermore, it improves the structure determination of the icosahedral capsid by dealing with the defocus gradient across the particle. LocalRec is expected to be widely applicable in a range of cryo-EM investigations of flexible and symmetry mismatched complexes.


Subject(s)
Adenoviridae/chemistry , Cryoelectron Microscopy/methods , Macromolecular Substances/chemistry , Viral Proteins/chemistry , Crystallography, X-Ray , Databases, Protein , Image Processing, Computer-Assisted , Models, Molecular , Protein Conformation , Protein Multimerization , Single Molecule Imaging
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