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,
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.
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,
. 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
.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude
. Heavily glycosylated S trimers bind to the ...angiotensin-converting enzyme 2 receptor and mediate entry of virions into target cells
. 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 membranes
. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy
, 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.
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
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
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
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
-3, where it replaces the existing particle-polishing method, as it outperforms the latter in all tests conducted.
Type A γ-aminobutyric acid receptors (GABA
A
Rs) are pentameric ligand-gated ion channels (pLGICs) and the main drivers of fast inhibitory neurotransmission in the vertebrate nervous system
1
,
2
. ...Their dysfunction is implicated in a range of neurological disorders, including depression, epilepsy and schizophrenia
3
,
4
. Amongst the numerous assemblies theoretically possible, α1β2/3γ2 GABA
A
Rs are most prevalent in the brain
5
. The β3 subunit plays an important role in maintaining inhibitory tone and expression of this subunit alone is sufficient to rescue inhibitory synaptic transmission in a CRISPR/Cas9 derived β1-3 triple knockout
6
. To date, efforts to generate accurate structural models for heteromeric GABA
A
Rs have been hampered by the use of engineered receptors and the presence of detergents
7
–
9
. Significantly, some recent cryo-EM reconstructions report “collapsed” conformations
8
,
9
which disagree with the prototypical pLGIC, the Torpedo nicotinic acetylcholine receptor
10
,
11
, the large body of structural work on homologous homopentameric receptor variants
12
, and the logic of a ion channel architecture. To address this problem, here we present a high-resolution cryo-EM structure of the full-length human α1β3γ2L, a major synaptic GABA
A
R isoform, functionally reconstituted in lipid nanodiscs. The receptor is bound to a positive allosteric modulator megabody and in a desensitised conformation. Unexpectedly, each GABA
A
R pentamer harbours two phosphatidylinositol 4,5-bisphosphate (PIP2) molecules, whose head groups 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 were not included. This structure illustrates the molecular principles of heteromeric GABA
A
receptor organization and provides a reference framework for future mechanistic investigations of GABA-ergic signalling and pharmacology.
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.
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.
Type-A γ-aminobutyric (GABA
) 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 GABA
receptors remains largely unknown. Here we report several high-resolution cryo-electron microscopy structures in which the full-length human α1β3γ2L GABA
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 GABA
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 GABA
receptor modulators.
Type-A γ-aminobutyric receptors (GABA
A
Rs) 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 common substances of abuse. Without reliable structural data, the mechanistic basis for pharmacological modulation of GABA
A
Rs remains largely unknown. Here we report high-resolution cryoEM structures of the full-length human α1β3γ2L GABA
A
R in lipid nanodiscs, bound to the channel blocker picrotoxin, the competitive antagonist bicuculline, the agonist GABA and the classical benzodiazepines alprazolam (Xanax) and diazepam (Valium), respectively. We describe the binding modes and mechanistic impacts of these ligands, the closed and desensitised states of the GABA
A
R gating cycle, and the basis for allosteric coupling between the extracellular, agonist-binding, and the transmembrane, pore-forming, regions. This work provides a structural framework to integrate decades of physiology and pharmacology research and a rational basis for development of novel GABA
A
R modulators.
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