Single-Particle Cryo-EM of Arabinofuranosyltransferase AftD from Mycobacteria, Wild-type
Ontology highlight
ABSTRACT:
SUBMITTER: Yong Zi Tan
PROVIDER: EMPIAR-10399 | biostudies-other |
REPOSITORIES: biostudies-other
Publications
Cryo-EM Structures and Regulation of Arabinofuranosyltransferase AftD from Mycobacteria.
Tan Yong Zi YZ Zhang Lei L Rodrigues José J Zheng Ruixiang Blake RB Giacometti Sabrina I SI Rosário Ana L AL Kloss Brian B Dandey Venkata P VP Wei Hui H Brunton Richard R Raczkowski Ashleigh M AM Athayde Diogo D Catalão Maria João MJ Pimentel Madalena M Clarke Oliver B OB Lowary Todd L TL Archer Margarida M Niederweis Michael M Potter Clinton S CS Carragher Bridget B Mancia Filippo F
Molecular cell 20200507 4
Mycobacterium tuberculosis causes tuberculosis, a disease that kills over 1 million people each year. Its cell envelope is a common antibiotic target and has a unique structure due, in part, to two lipidated polysaccharides-arabinogalactan and lipoarabinomannan. Arabinofuranosyltransferase D (AftD) is an essential enzyme involved in assembling these glycolipids. We present the 2.9-Å resolution structure of M. abscessus AftD, determined by single-particle cryo-electron microscopy. AftD has a cons ...[more]
Similar Datasets
Project description:Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.
| S-EPMC6009202 | biostudies-literature
Project description:The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years1,2. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABAA receptor homopentamer3. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.
| S-EPMC7611073 | biostudies-literature
Project description: Not available
| EMPIAR-10391 | biostudies-other
Project description:Frealign is a software tool designed to process electron microscope images of single molecules and complexes to obtain reconstructions at the highest possible resolution. It provides a number of refinement parameters and options that allow users to tune their refinement to achieve specific goals, such as masking to classify selected regions within a particle, control over the refinement of specific alignment parameters to accommodate various data collection schemes, refinement of pseudosymmetric particles, and generation of initial maps. This chapter provides a general overview of Frealign functions and a more detailed guide to using Frealign in typical scenarios.
| S-EPMC6760665 | biostudies-literature
Project description:JSPR is a single particle cryo-EM image processing and 3D reconstruction software developed in the Jiang laboratory at Purdue University. It began as a few refinement scripts for symmetric and asymmetric reconstructions of icosahedral viruses, but has grown into a comprehensive suite of tools for building ab initio reconstructions, high resolution refinements of viruses, protein complexes of arbitrary symmetries including helical tubes/filaments, and image file handling utilities. In this review, we will present examples achieved using JSPR and demonstrate recently implemented features of JSPR such as multi-aberration "alignments" and automatic optimization of masking for the assessment of map resolution using "true" FSC.
| S-EPMC7779749 | biostudies-literature
Project description:Preferred particle orientation represents a recurring problem in single-particle cryogenic electron microcopy (cryo-EM). A specimen-independent approach through tilting has been attempted to increase particle orientation coverage, thus minimizing anisotropic three-dimensional (3D) reconstruction. However, focus gradient is a critical issue hindering tilt applications from being a general practice in single-particle cryo-EM. The present study describes a newly developed geometrically optimized approach, goCTF, to reliably determine the global focus gradient. A novel strategy of determining contrast transfer function (CTF) parameters from a sector of the signal preserved power spectrum is applied to increase reliability. Subsequently, per-particle based local focus refinement is conducted in an iterative manner to further improve the defocus accuracy. Novel diagnosis methods using a standard deviation defocus plot and goodness of fit heatmap have also been proposed to evaluate CTF fitting quality prior to 3D refinement. In a benchmark study, goCTF processed a published single-particle cryo-EM dataset for influenza hemagglutinin trimer collected at a 40-degree specimen tilt. The resulting 3D reconstruction map was improved from 4.1 Å to 3.7 Å resolution. The goCTF program is built on the open-source code of CTFFIND4, which adopts a consistent user interface for ease of use.
| S-EPMC6692911 | biostudies-literature
Project description:We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional defocus approach. Spherical aberration becomes a limiting factor for achieving resolutions below 3 Å with in-focus phase plate images. The phase plate could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach.
| S-EPMC4850076 | biostudies-literature
Project description:Single-particle cryogenic electron microscopy has recently become a major method for determining the structures of proteins and protein complexes. This has markedly increased the demand for throughput of high-resolution electron microscopes, which are required to produce high-resolution images at high rates. An increase in data-collection throughput can be achieved by using large beam-image shifts combined with off-axis coma correction, enabling the acquisition of multiple images from a large area of the EM grid without moving the microscope stage. Here, the optical properties of the JEOL CRYO ARM 300 electron microscope equipped with a K3 camera were characterized under off-axis illumination conditions. It is shown that efficient coma correction can be achieved for beam-image shifts with an amplitude of at least 10 µm, enabling a routine throughput for data collection of between 6000 and 9000 images per day. Use of the benchmark for the rapid data-collection procedure (with beam-image shifts of up to 7 µm) on apoferritin resulted in a reconstruction at a resolution of 1.7 Å. This demonstrates that the rapid automated acquisition of high-resolution micrographs is possible using a CRYO ARM 300.
| S-EPMC8098478 | biostudies-literature
Project description:The combination of high-end cryogenic transmission electron microscopes (cryo-EM), direct electron detectors, and advanced image algorithms allows researchers to obtain the 3D structures of much smaller macromolecules than years ago. However, there are still major challenges for the single-particle cryo-EM method to achieve routine structure determinations for macromolecules much smaller than 100 kDa, which are the majority of all plant and animal proteins. These challenges include sample characteristics such as sample heterogeneity, beam damage, ice layer thickness, stability, and quality, as well as hardware limitations such as detector performance, beam, and phase plate quality. Here, single particle data sets were simulated for samples that were ideal in terms of homogeneity, distribution, and stability, but with realistic parameters for ice layer, dose, detector performance, and beam characteristics. Reference data were calculated for human apo-ferritin using identical parameters reported for an experimental data set downloaded from EMPIAR. Processing of the simulated data set resulted in a value of 1.86 Å from 20?214 particles, similar to a 2 Å density map obtained from 29?224 particles selected from real micrographs. Simulated data sets were then generated for a 14 kDa protein, hen egg white lysozyme (HEWL), with and without an ideal phase plate (PP). Whereas we could not obtain a high-resolution 3D reconstruction of HEWL for the data set without PP, the one with PP resulted in a 2.78 Å resolution density map from 225?751 particles. Our simulator and simulations could help in pushing the size limits of cryo-EM.
| S-EPMC7254834 | biostudies-literature
Project description:We present a strategy for tackling preferred specimen orientation in single-particle cryogenic electron microscopy by employing tilts during data collection. We also describe a tool to quantify the resulting directional resolution using 3D Fourier shell correlation volumes. We applied these methods to determine the structures at near-atomic resolution of the influenza hemagglutinin trimer, which adopts a highly preferred specimen orientation, and of ribosomal biogenesis intermediates, which adopt moderately preferred orientations.
| S-EPMC5533649 | biostudies-literature