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Electron tomography of frozen, hydrated samples allows structure determination of macromolecular complexes that are embedded in complex environments. Provided that the target complexes may be localised in noisy, three-dimensional tomographic reconstructions, averaging images of multiple instances of these molecules can lead to structures with sufficient resolution for de novo atomic modelling. Although many research groups have contributed image processing tools for these tasks, a lack of standardisation and interoperability represents a barrier for newcomers to the field. Here, we present an image processing pipeline for electron tomography data in RELION-5, with functionality ranging from the import of unprocessed movies to the automated building of atomic models in the final maps. Our explicit definition of metadata items that describe the steps of our pipeline has been designed for interoperability with other software tools and provides a framework for further standardisation.
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We present a new approach for macromolecular structure determination from multiple particles in electron cryo-tomography (cryo-ET) data sets. Whereas existing subtomogram averaging approaches are based on 3D data models, we propose to optimise a regularised likelihood target that approximates a function of the 2D experimental images. In addition, analogous to Bayesian polishing and contrast transfer function (CTF) refinement in single-particle analysis, we describe the approaches that exploit the increased signal-to-noise ratio in the averaged structure to optimise tilt-series alignments, beam-induced motions of the particles throughout the tilt-series acquisition, defoci of the individual particles, as well as higher-order optical aberrations of the microscope. Implementation of our approaches in the open-source software package RELION aims to facilitate their general use, particularly for those researchers who are already familiar with its single-particle analysis tools. We illustrate for three applications that our approaches allow structure determination from cryo-ET data to resolutions sufficient for de novo atomic modelling.
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Elétrons , Processamento de Imagem Assistida por Computador , Processamento de Imagem Assistida por Computador/métodos , Teorema de Bayes , Microscopia Crioeletrônica/métodos , Tomografia com Microscopia Eletrônica/métodosRESUMO
A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19845-z .
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Urease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. The obtained structure is of sufficient quality to support drug development efforts.
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Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Urease/química , Urease/metabolismo , Yersinia enterocolitica/enzimologia , Domínio Catalítico , Microscopia Crioeletrônica , Lisina/metabolismo , Modelos Moleculares , Níquel/química , Níquel/metabolismo , Conformação Proteica , Domínios Proteicos , Água/químicaRESUMO
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude1. Heavily glycosylated S trimers bind to the angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells2-6. S exhibits extensive conformational flexibility: it modulates exposure of its receptor-binding site and subsequently undergoes complete structural rearrangement to drive fusion of viral and cellular membranes2,7,8. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy2,7,9-12, but the structure and distribution of S on the virion surface remain unknown. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions and determine the high-resolution structure, conformational flexibility and distribution of S trimers in situ on the virion surface. These results reveal the conformations of S on the virion, and provide a basis from which to understand interactions between S and neutralizing antibodies during infection or vaccination.
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Microscopia Crioeletrônica , SARS-CoV-2/metabolismo , SARS-CoV-2/ultraestrutura , Glicoproteína da Espícula de Coronavírus/análise , Glicoproteína da Espícula de Coronavírus/ultraestrutura , Vírion/química , Vírion/ultraestrutura , Anticorpos Neutralizantes/imunologia , COVID-19/imunologia , Vacinas contra COVID-19/imunologia , Linhagem Celular Tumoral , Humanos , Modelos Moleculares , Maleabilidade , Conformação Proteica , Multimerização Proteica , SARS-CoV-2/química , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/isolamento & purificação , Vírion/isolamento & purificação , Vírion/metabolismoRESUMO
Methods are presented that detect three types of aberrations in single-particle cryo-EM data sets: symmetrical and antisymmetrical optical aberrations and magnification anisotropy. Because these methods only depend on the availability of a preliminary 3D reconstruction from the data, they can be used to correct for these aberrations for any given cryo-EM data set, a posteriori. Using five publicly available data sets, it is shown that considering these aberrations improves the resolution of the 3D reconstruction when these effects are present. The methods are implemented in version 3.1 of the open-source software package RELION.
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Chronic traumatic encephalopathy (CTE) is a neurodegenerative tauopathy that is associated with repetitive head impacts or exposure to blast waves. First described as punch-drunk syndrome and dementia pugilistica in retired boxers1-3, CTE has since been identified in former participants of other contact sports, ex-military personnel and after physical abuse4-7. No disease-modifying therapies currently exist, and diagnosis requires an autopsy. CTE is defined by an abundance of hyperphosphorylated tau protein in neurons, astrocytes and cell processes around blood vessels8,9. This, together with the accumulation of tau inclusions in cortical layers II and III, distinguishes CTE from Alzheimer's disease and other tauopathies10,11. However, the morphologies of tau filaments in CTE and the mechanisms by which brain trauma can lead to their formation are unknown. Here we determine the structures of tau filaments from the brains of three individuals with CTE at resolutions down to 2.3 Å, using cryo-electron microscopy. We show that filament structures are identical in the three cases but are distinct from those of Alzheimer's and Pick's diseases, and from those formed in vitro12-15. Similar to Alzheimer's disease12,14,16-18, all six brain tau isoforms assemble into filaments in CTE, and residues K274-R379 of three-repeat tau and S305-R379 of four-repeat tau form the ordered core of two identical C-shaped protofilaments. However, a different conformation of the ß-helix region creates a hydrophobic cavity that is absent in tau filaments from the brains of patients with Alzheimer's disease. This cavity encloses an additional density that is not connected to tau, which suggests that the incorporation of cofactors may have a role in tau aggregation in CTE. Moreover, filaments in CTE have distinct protofilament interfaces to those of Alzheimer's disease. Our structures provide a unifying neuropathological criterion for CTE, and support the hypothesis that the formation and propagation of distinct conformers of assembled tau underlie different neurodegenerative diseases.
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Encefalopatia Traumática Crônica , Microscopia Crioeletrônica , Interações Hidrofóbicas e Hidrofílicas , Dobramento de Proteína , Proteínas tau/química , Proteínas tau/ultraestrutura , Idoso , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Encefalopatia Traumática Crônica/metabolismo , Encefalopatia Traumática Crônica/patologia , Lobo Frontal/metabolismo , Lobo Frontal/patologia , Humanos , Masculino , Modelos MolecularesRESUMO
A new method to estimate the trajectories of particle motion and the amount of cumulative beam damage in electron cryo-microscopy (cryo-EM) single-particle analysis is presented. The motion within the sample is modelled through the use of Gaussian process regression. This allows a prior likelihood that favours spatially and temporally smooth motion to be associated with each hypothetical set of particle trajectories without imposing hard constraints. This formulation enables the a posteriori likelihood of a set of particle trajectories to be expressed as a product of that prior likelihood and an observation likelihood given by the data, and this a posteriori likelihood to then be maximized. Since the smoothness prior requires three parameters that describe the statistics of the observed motion, an efficient stochastic method to estimate these parameters is also proposed. Finally, a practical algorithm is proposed that estimates the average amount of cumulative radiation damage as a function of radiation dose and spatial frequency, and then fits relative B factors to that damage in a robust way. The method is evaluated on three publicly available data sets, and its usefulness is illustrated by comparison with state-of-the-art methods and previously published results. The new method has been implemented as Bayesian polishing in RELION-3, where it replaces the existing particle-polishing method, as it outperforms the latter in all tests conducted.
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In Fig. 5b, d, the arrows showing transmembrane domain rotations were inadvertently pointing clockwise instead of anticlockwise. Similarly, 'anticlockwise' should have been 'clockwise' in the sentence 'This conformational change of the ECD triggers a clockwise rotation of the TMD.' In Extended Data Table 1, the units of the column 'Model resolution' should have been Å instead of Å2. These errors have been corrected online.
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Type-A γ-aminobutyric (GABAA) receptors are ligand-gated chloride channels with a very rich pharmacology. Some of their modulators, including benzodiazepines and general anaesthetics, are among the most successful drugs in clinical use and are common substances of abuse. Without reliable structural data, the mechanistic basis for the pharmacological modulation of GABAA receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1ß3γ2L GABAA receptor in lipid nanodiscs is bound to the channel-blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA (γ-aminobutyric acid), and the classical benzodiazepines alprazolam and diazepam. We describe the binding modes and mechanistic effects of these ligands, the closed and desensitized states of the GABAA receptor gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding region and the transmembrane, pore-forming region. This work provides a structural framework in which to integrate previous physiology and pharmacology research and a rational basis for the development of GABAA receptor modulators.
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Alprazolam/química , Bicuculina/química , Microscopia Crioeletrônica , Diazepam/química , Picrotoxina/química , Receptores de GABA-A/química , Receptores de GABA-A/metabolismo , Transdução de Sinais/efeitos dos fármacos , Regulação Alostérica/efeitos dos fármacos , Alprazolam/farmacologia , Benzodiazepinas/química , Benzodiazepinas/farmacologia , Bicuculina/farmacologia , Ligação Competitiva/efeitos dos fármacos , Diazepam/farmacologia , Moduladores GABAérgicos/química , Moduladores GABAérgicos/farmacologia , Humanos , Ligantes , Modelos Moleculares , Nanoestruturas/química , Picrotoxina/farmacologiaRESUMO
Type A γ-aminobutyric acid (GABAA) receptors are pentameric ligand-gated ion channels and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system1,2. Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia3,4. Among the numerous assemblies that are theoretically possible, the most prevalent in the brain are the α1ß2/3γ2 GABAA receptors5. The ß3 subunit has an important role in maintaining inhibitory tone, and the expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in ß1-ß3 triple knockout neurons6. So far, efforts to generate accurate structural models for heteromeric GABAA receptors have been hampered by the use of engineered receptors and the presence of detergents7-9. Notably, some recent cryo-electron microscopy reconstructions have reported 'collapsed' conformations8,9; however, these disagree with the structure of the prototypical pentameric ligand-gated ion channel the Torpedo nicotinic acetylcholine receptor10,11, the large body of structural work on homologous homopentameric receptor variants12 and the logic of an ion-channel architecture. Here we present a high-resolution cryo-electron microscopy structure of the full-length human α1ß3γ2L-a major synaptic GABAA receptor isoform-that is functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator 'megabody' and is in a desensitized conformation. Each GABAA receptor pentamer contains two phosphatidylinositol-4,5-bisphosphate molecules, the head groups of which occupy positively charged pockets in the intracellular juxtamembrane regions of α1 subunits. Beyond this level, the intracellular M3-M4 loops are largely disordered, possibly because interacting post-synaptic proteins are not present. This structure illustrates the molecular principles of heteromeric GABAA receptor organization and provides a reference framework for future mechanistic investigations of GABAergic signalling and pharmacology.
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Microscopia Crioeletrônica , Bicamadas Lipídicas/química , Receptores de GABA-A/química , Receptores de GABA-A/ultraestrutura , Regulação Alostérica , Sequência de Aminoácidos , Sítios de Ligação , Condutividade Elétrica , Humanos , Modelos Moleculares , Simulação de Acoplamento Molecular , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/ultraestrutura , Estrutura Quaternária de Proteína , Receptores de GABA-A/metabolismoRESUMO
Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 Å compared to previous RELION versions.