Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution

Electron cryo-microscopy (cryo-EM) is becoming one of the routine tools for structure determination of biological macromolecules. Commonly, molecular images are obtained by conventional transmission electron microcopy (CTEM) using underfocus and subsequently computationally combined into a high-resolution 3D structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to the cryo-EM test specimen of tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination at 3.5 Angstrom resolution using single-particle based helical reconstruction. A series of cryo-EM TMV maps was resolved at near-atomic resolution taken at different convergence semi-angle (CSA) beams and share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. The associated map B-factors from iDPC-STEM match those obtained by CTEM recordings using 2nd generation direct electron detection devices. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.

Structural and functional characterization of the intracellular filament-forming nitrite oxidoreductase multiprotein complex

Nitrate is an abundant nutrient and electron acceptor throughout Earth’s biosphere. Virtually all nitrate in nature is produced by the oxidation of nitrite by the nitrite oxidoreductase (NXR) multiprotein complex. NXR is a crucial enzyme in the global biological nitrogen cycle, and is found in nitrite-oxidizing bacteria (including comammox organisms), which generate the bulk of the nitrate in the environment, and in anaerobic ammonium-oxidizing (anammox) bacteria which produce half of the dinitrogen gas in our atmosphere. However, despite its central role in biology and decades of intense study, no structural information on NXR is available. Here, we present a structural and biochemical analysis of the NXR from the anammox bacterium Kuenenia stuttgartiensis, integrating X-ray crystallography, cryo-electron tomography, helical reconstruction cryo-electron microscopy, interaction and reconstitution studies and enzyme kinetics. We find that NXR catalyses both nitrite oxidation and nitrate reduction, and show that in the cell, NXR is arranged in tubules several hundred nanometres long. We reveal the tubule architecture and show that tubule formation is induced by a previously unidentified, haem-containing subunit, NXR-T. The results also reveal unexpected features in the active site of the enzyme, an unusual cofactor coordination in the protein’s electron transport chain, and elucidate the electron transfer pathways within the complex.

Common computed tomography artifact: source and avoidance

Background Artifacts have significantly degraded the quality of computed tomography (CT) images, to the extent of making them unusable for diagnosis. The types of artifact that could be used are as follows: (a) streaking, which is commonly due to a discrepancy in a single measurement, (b) shading, which is due to a group of channels deviating gradually from the true measurement, (c) rings, which are due to errors in individual detector calibration and (d) distortion, which is due to helical reconstruction. It is occasionally possible to avoid scanning of a bony area, by means of changing the postion of the patient. Thus, this study aimed to evaluate the common artifacts that affect image quality and the method of correction to improve image quality. Results The data were collected by distributing a questionnaire to the CT technologist at different hospitals about the most common type of artifacts in the CT images, source of artifacts and methods of correction. A total of 95 CT technologists responded to the questionnaire, which included 67% males and 33% females. Most of the participants (70%) were experienced CT technologists, and 61% of the participants had not done any subspecialty CT scan courses. The most common artifact used in the CT departments was motion artifact in brain CT (73%), and the best method to reduce motion artifact was patient preparation (87%). Conclusions The most common shown artifact in this study was motion artifact, and the common cause was the patient-based artifact. It is important to understand why objects occur and how they could be prevented or suppressed to improve image quality.

Bacillus endospore appendages form a novel family of disulfide-linked pili

SummaryBacillus cereus sensu lato is a group of Gram-positive endospore-forming bacteria with high ecological diversity. Their endospores are decorated with micrometer-long appendages of unknown identity and function. Here we isolate endospore appendages (Enas) from the food poisoning outbreak strain B. cereus NVH 0075-95 and find proteinaceous fibers of two main morphologies. By using cryo-EM and 3D helical reconstruction we show that Bacillus Enas form a novel class of Gram-positive pili. Enas consist of single domain subunits with jellyroll topology that are laterally stacked by β-sheet augmentation. Enas are longitudinally stabilized by disulfide bonding through N-terminal connector peptides that bridge the helical turns. Together, this results in flexible pili that are highly resistant to heat, drought and chemical damage. Phylogenomic analysis reveals the presence of defined ena clades amongst different eco- and pathotypes. We propose Enas to represent a novel class of pili specifically adapted to the harsh conditions encountered by bacterial spores.

Amyloid structure determination in RELION-3.1

Helical reconstruction in RELION is increasingly being used to determine the atomic structures of amyloid filaments from electron cryo-microscopy (cryo-EM) images. However, because the energy landscape of amyloid refinements is typically fraught with local optima, amyloid structure determination is often difficult. This paper aims to help RELION users in this process. It discusses aspects of helical reconstruction that are particularly relevant to amyloids, it illustrates the problem of local optima in refinement and how to detect them, and it introduces a new method to calculate 3D initial models from reference-free 2D class averages. By providing starting models that are closer to the global optimum, this method makes amyloid structure determination easier. All methods described are open-source and distributed within RELION-3.1. Their use is illustrated using a publicly available data set on tau filaments from the brain of an individual with Alzheimer's disease.

Amyloid structure determination in RELION-3.1

Helical reconstruction in RELION is increasingly used to determine atomic structures of amyloid filaments from electron cryo-microscopy (cryo-EM) images. However, because the energy landscape of amyloid refinements is typically fraught with local optima, amyloid structure determination is often difficult. This paper aims to help RELION users in this process. It discusses aspects of helical reconstruction that are specific to amyloids; it illustrates the problem of local optima in refinement and how to detect these; and it introduces a new method to calculate 3D initial models from reference-free 2D class averages. By providing starting models that are closer to the global optimum, this method makes amyloid structure determination easier. All methods described are open-source and distributed within RELION-3.1. Their use is illustrated using a publicly available data set on tau filaments from the brain of an individual with Alzheimer’s disease.