Direct IgG epitope mapping on bacterial AB toxins by cryo-EM.
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ABSTRACT:
SUBMITTER: Nguyen T
PROVIDER: S-EPMC8496303 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Background and objectiveThe contrast of cryo-EM images varies from one to another, primarily due to the uneven thickness of the ice layer. This contrast variation can affect the quality of 2-D class averaging, 3-D ab-initio modeling, and 3-D heterogeneity analysis. Contrast estimation is currently performed during 3-D iterative refinement. As a result, the estimates are not available at the earlier computational stages of class averaging and ab-initio modeling. This paper aims to solve the contrast estimation problem directly from the picked particle images in the ab-initio stage, without estimating the 3-D volume, image rotations, or class averages.MethodsThe key observation underlying our analysis is that the 2-D covariance matrix of the raw images is related to the covariance of the underlying clean images, the noise variance, and the contrast variability between images. We show that the contrast variability can be derived from the 2-D covariance matrix and we apply the existing Covariance Wiener Filtering (CWF) framework to estimate it. We also demonstrate a modification of CWF to estimate the contrast of individual images.ResultsOur method improves the contrast estimation by a large margin, compared to the previous CWF method. Its estimation accuracy is often comparable to that of an oracle that knows the ground truth covariance of the clean images. The more accurate contrast estimation also improves the quality of image restoration as demonstrated in both synthetic and experimental datasets.ConclusionsThis paper proposes an effective method for contrast estimation directly from noisy images without using any 3-D volume information. It enables contrast correction in the earlier stage of single particle analysis, and may improve the accuracy of downstream processing.
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Project description:Cryogenic electron microscopy (cryo-EM) and single-particle analysis now enables the determination of high-resolution structures of macromolecular assemblies that have resisted X-ray crystallography and other approaches. We developed the SIMPLE open-source image-processing suite for analysing cryo-EM images of single-particles. A core component of SIMPLE is the probabilistic PRIME algorithm for identifying clusters of images in 2D and determine relative orientations of single-particle projections in 3D. Here, we extend our previous work on PRIME and introduce new stochastic optimization algorithms that improve the robustness of the approach. Our refined method for identification of homogeneous subsets of images in accurate register substantially improves the resolution of the cluster centers and of the ab initio 3D reconstructions derived from them. We now obtain maps with a resolution better than 10 Å by exclusively processing cluster centers. Excellent parallel code performance on over-the-counter laptops and CPU workstations is demonstrated.
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Project description:Protein nanocompartments are widespread in bacteria and archaea, but their functions are not yet well understood. Here, the cryo-EM structure of a nanocompartment from the thermophilic bacterium Thermotoga maritima is reported at 2.0 Å resolution. The high resolution of this structure shows that interactions in the E-loop domain may be important for the thermostability of the nanocompartment assembly. Also, the channels at the fivefold axis, threefold axis and dimer interface are assessed for their ability to transport iron. Finally, an unexpected flavin ligand was identified on the exterior of the shell, indicating that this nanocompartment may also play a direct role in iron metabolism.
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Project description:Transcription is the principal control point for bacterial gene expression, and it enables a global cellular response to an intracellular or environmental trigger. Transcriptional regulation is orchestrated by transcription factors, which activate or repress transcription of target genes by modulating the activity of RNA polymerase. Dissecting the nature and precise choreography of these interactions is essential for developing a molecular understanding of transcriptional regulation. While the contribution of X-ray crystallography has been invaluable, the 'resolution revolution' of cryo-electron microscopy has transformed our structural investigations, enabling large, dynamic and often transient transcription complexes to be resolved that in many cases had resisted crystallisation. In this review, we highlight the impact cryo-electron microscopy has had in gaining a deeper understanding of transcriptional regulation in bacteria. We also provide readers working within the field with an overview of the recent innovations available for cryo-electron microscopy sample preparation and image reconstruction of transcription complexes.
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Project description:Cryo-Electron Microscopy (cryo-EM) is a widely used and effective method for determining the three-dimensional (3D) structure of biological molecules. For ab-initio Cryo-EM 3D reconstruction using single particle analysis (SPA), estimating the projection direction of the projection image is a crucial step. However, the existing SPA methods based on common lines are sensitive to noise. The error in common line detection will lead to a poor estimation of the projection directions and thus may greatly affect the final reconstruction results. To improve the reconstruction results, multiple candidate common lines are estimated for each pair of projection images. The key problem then becomes a combination optimization problem of selecting consistent common lines from multiple candidates. To solve the problem efficiently, a physics-inspired method based on a kinetic model is proposed in this work. More specifically, hypothetical attractive forces between each pair of candidate common lines are used to calculate a hypothetical torque exerted on each projection image in the 3D reconstruction space, and the rotation under the hypothetical torque is used to optimize the projection direction estimation of the projection image. This way, the consistent common lines along with the projection directions can be found directly without enumeration of all the combinations of the multiple candidate common lines. Compared with the traditional methods, the proposed method is shown to be able to produce more accurate 3D reconstruction results from high noise projection images. Besides the practical value, the proposed method also serves as a good reference for solving similar combinatorial optimization problems.
| S-EPMC10805181 | biostudies-literature
Project description:Stimulator of interferon genes (STING) is an antiviral signalling protein that is broadly conserved in both innate immunity in animals and phage defence in prokaryotes1-4. Activation of STING requires its assembly into an oligomeric filament structure through binding of a cyclic dinucleotide4-13, but the molecular basis of STING filament assembly and extension remains unknown. Here we use cryogenic electron microscopy to determine the structure of the active Toll/interleukin-1 receptor (TIR)-STING filament complex from a Sphingobacterium faecium cyclic-oligonucleotide-based antiphage signalling system (CBASS) defence operon. Bacterial TIR-STING filament formation is driven by STING interfaces that become exposed on high-affinity recognition of the cognate cyclic dinucleotide signal c-di-GMP. Repeating dimeric STING units stack laterally head-to-head through surface interfaces, which are also essential for human STING tetramer formation and downstream immune signalling in mammals5. The active bacterial TIR-STING structure reveals further cross-filament contacts that brace the assembly and coordinate packing of the associated TIR NADase effector domains at the base of the filament to drive NAD+ hydrolysis. STING interface and cross-filament contacts are essential for cell growth arrest in vivo and reveal a stepwise mechanism of activation whereby STING filament assembly is required for subsequent effector activation. Our results define the structural basis of STING filament formation in prokaryotic antiviral signalling.
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Project description:Phytochromes are red-light photoreceptors discovered in plants with homologs in bacteria and fungi that regulate a variety of physiological responses. They display a reversible photocycle between two distinct states: a red-light-absorbing Pr state and a far-red light-absorbing Pfr state. The photoconversion regulates the activity of an enzymatic domain, usually a histidine kinase (HK). The molecular mechanism that explains how light controls the HK activity is not understood because structures of unmodified bacterial phytochromes with HK activity are missing. Here, we report three cryo-electron microscopy structures of a wild-type bacterial phytochrome with HK activity determined as Pr and Pfr homodimers and as a Pr/Pfr heterodimer with individual subunits in distinct states. We propose that the Pr/Pfr heterodimer is a physiologically relevant signal transduction intermediate. Our results offer insight into the molecular mechanism that controls the enzymatic activity of the HK as part of a bacterial two-component system that perceives and transduces light signals.
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Project description:The initiation of bacterial translation involves the tightly regulated joining of the 50S ribosomal subunit to an initiator transfer RNA (fMet-tRNAfMet)-containing 30S ribosomal initiation complex to form a 70S initiation complex, which subsequently matures into a 70S elongation-competent complex. Rapid and accurate formation of the 70S initiation complex is promoted by initiation factors, which must dissociate from the 30S initiation complex before the resulting 70S elongation-competent complex can begin the elongation of translation1. Although comparisons of the structures of the 30S2-5 and 70S4,6-8 initiation complexes have revealed that the ribosome, initiation factors and fMet-tRNAfMet can acquire different conformations in these complexes, the timing of conformational changes during formation of the 70S initiation complex, the structures of any intermediates formed during these rearrangements, and the contributions that these dynamics might make to the mechanism and regulation of initiation remain unknown. Moreover, the absence of a structure of the 70S elongation-competent complex formed via an initiation-factor-catalysed reaction has precluded an understanding of the rearrangements to the ribosome, initiation factors and fMet-tRNAfMet that occur during maturation of a 70S initiation complex into a 70S elongation-competent complex. Here, using time-resolved cryogenic electron microscopy9, we report the near-atomic-resolution view of how a time-ordered series of conformational changes drive and regulate subunit joining, initiation factor dissociation and fMet-tRNAfMet positioning during formation of the 70S elongation-competent complex. Our results demonstrate the power of time-resolved cryogenic electron microscopy to determine how a time-ordered series of conformational changes contribute to the mechanism and regulation of one of the most fundamental processes in biology.
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Project description:The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca(2+) release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the multiple orientations of the antibody bound in the tetrameric RyR1, were overcome by modifying the 3D reconstruction scheme. This approach enabled us to ascertain that the three antibodies bind to the same region, to obtain a 3D reconstruction of RyR1 with the antibody bound, and to map SPRY2 to the periphery of the cytoplasmic domain of RyR1. We report here the first 3D localization of a SPRY2 domain in any known RyR isoform.
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Project description:Our understanding of protein synthesis has been conceptualised around the structure and function of the bacterial ribosome. This complex macromolecular machine is the target of important antimicrobial drugs, an integral line of defence against infectious diseases. Here, we describe how open access to cryo-electron microscopy facilities combined with bespoke user support enabled structural determination of the translating ribosome from Escherichia coli at 1.55 Å resolution. The obtained structures allow for direct determination of the rRNA sequence to identify ribosome polymorphism sites in the E. coli strain used in this study and enable interpretation of the ribosomal active and peripheral sites at unprecedented resolution. This includes scarcely populated chimeric hybrid states of the ribosome engaged in several tRNA translocation steps resolved at ~2 Å resolution. The current map not only improves our understanding of protein synthesis but also allows for more precise structure-based drug design of antibiotics to tackle rising bacterial resistance.
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