Tomo Live: an on-the-fly reconstruction pipeline to judge data quality for cryo-electron tomography workflows.

Data acquisition and processing for cryo-electron tomography can be a significant bottleneck for users. To simplify and streamline the cryo-ET workflow, Tomo Live, an on-the-fly solution that automates the alignment and reconstruction of tilt-series data, enabling real-time data-quality assessment, has been developed. Through the integration of Tomo Live into the data-acquisition workflow for cryo-ET, motion correction is performed directly after each of the acquired tilt angles.

Cryo-electron tomography of intact cardiac muscle reveals myosin binding protein-C linking myosin and actin filaments.

Myosin binding protein C (MyBP-C) is an accessory protein of the thick filament in vertebrate cardiac muscle arranged over 9 stripes of intervals of 430 Å in each half of the A-band in the region called the C-zone. Mutations in cardiac MyBP-C are a leading cause of hypertrophic cardiomyopathy the mechanism of which is unknown. It is a rod-shaped protein composed of 10 or 11 immunoglobulin- or fibronectin-like domains labelled C0 to C10 which binds to the thick filament via its C-terminal region.

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 Å resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter.

MamY is a membrane-bound protein that aligns magnetosomes and the motility axis of helical magnetotactic bacteria.

To navigate within the geomagnetic field, magnetotactic bacteria synthesize magnetosomes, which are unique organelles consisting of membrane-enveloped magnetite nanocrystals. In magnetotactic spirilla, magnetosomes become actively organized into chains by the filament-forming actin-like MamK and the adaptor protein MamJ, thereby assembling a magnetic dipole much like a compass needle. However, in Magnetospirillum gryphiswaldense, discontinuous chains are still formed in the absence of MamK.

Preparation of Bacterial Magnetosomes for Proteome Analysis.

Magnetotactic bacteria form unique prokaryotic organelles, termed magnetosomes, which consist of membrane-enclosed magnetite nanoparticles. Analysis of magnetosome biogenesis has been greatly facilitated by proteomic methods. These, however, require pure, highly enriched magnetosomes.

A quantitative assessment of the membrane-integral sub-proteome of a bacterial magnetic organelle.

Unlabelled: Magnetotactic bacteria produce chains of complex membrane-bound organelles that direct the biomineralization of magnetic nanoparticles and serve for magnetic field navigation. These magnetosome compartments have recently emerged as a model for studying the subcellular organization of prokaryotic organelles. Previous studies indicated the presence of specific proteins with various functions in magnetosome biosynthesis.

Genetic and Ultrastructural Analysis Reveals the Key Players and Initial Steps of Bacterial Magnetosome Membrane Biogenesis.

Magnetosomes of magnetotactic bacteria contain well-ordered nanocrystals for magnetic navigation and have recently emerged as the most sophisticated model system to study the formation of membrane bounded organelles in prokaryotes. Magnetosome biosynthesis is thought to begin with the formation of a dedicated compartment, the magnetosome membrane (MM), in which the biosynthesis of a magnetic mineral is strictly controlled. While the biomineralization of magnetosomes and their subsequent assembly into linear chains recently have become increasingly well studied, the molecular mechanisms and early stages involved in MM formation remained poorly understood.

Overproduction of Magnetosomes by Genomic Amplification of Biosynthesis-Related Gene Clusters in a Magnetotactic Bacterium.

Unlabelled: Magnetotactic bacteria biosynthesize specific organelles, the magnetosomes, which are membrane-enclosed crystals of a magnetic iron mineral that are aligned in a linear chain. The number and size of magnetosome particles have to be critically controlled to build a sensor sufficiently strong to ensure the efficient alignment of cells within Earth's weak magnetic field while at the same time minimizing the metabolic costs imposed by excessive magnetosome biosynthesis. Apart from their biological function, bacterial magnetosomes have gained considerable interest since they provide a highly useful model for prokaryotic organelle formation and represent biogenic magnetic nanoparticles with exceptional properties.

Bacterial magnetosome biomineralization--a novel platform to study molecular mechanisms of human CDF-related Type-II diabetes.

Cation diffusion facilitators (CDF) are part of a highly conserved protein family that maintains cellular divalent cation homeostasis in all organisms. CDFs were found to be involved in numerous human health conditions, such as Type-II diabetes and neurodegenerative diseases. In this work, we established the magnetite biomineralizing alphaproteobacterium Magnetospirillum gryphiswaldense as an effective model system to study CDF-related Type-II diabetes.

Genetic dissection of the mamAB and mms6 operons reveals a gene set essential for magnetosome biogenesis in Magnetospirillum gryphiswaldense.

Biosynthesis of bacterial magnetosomes, which are intracellular membrane-enclosed, nanosized magnetic crystals, is controlled by a set of >30 specific genes. In Magnetospirillum gryphiswaldense, these are clustered mostly within a large conserved genomic magnetosome island (MAI) comprising the mms6, mamGFDC, mamAB, and mamXY operons. Here, we demonstrate that the five previously uncharacterized genes of the mms6 operon have crucial functions in the regulation of magnetosome biomineralization that partially overlap MamF and other proteins encoded by the adjacent mamGFDC operon.

A tailored galK counterselection system for efficient markerless gene deletion and chromosomal tagging in Magnetospirillum gryphiswaldense.

Magnetotactic bacteria have emerged as excellent model systems to study bacterial cell biology, biomineralization, vesicle formation, and protein targeting because of their ability to synthesize single-domain magnetite crystals within unique organelles (magnetosomes). However, only few species are amenable to genetic manipulation, and the limited methods for site-specific mutagenesis are tedious and time-consuming. Here, we report the adaptation and application of a fast and convenient technique for markerless chromosomal manipulation of Magnetospirillum gryphiswaldense using a single antibiotic resistance cassette and galK-based counterselection for marker recycling.

The terminal oxidase cbb3 functions in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense.

The biomineralization of magnetosomes in Magnetospirillum gryphiswaldense and other magnetotactic bacteria occurs only under suboxic conditions. However, the mechanism of oxygen regulation and redox control of biosynthesis of the mixed-valence iron oxide magnetite [FeII(FeIII)2O4] is still unclear. Here, we set out to investigate the role of aerobic respiration in both energy metabolism and magnetite biomineralization of M.

Biosynthesis of magnetic nanostructures in a foreign organism by transfer of bacterial magnetosome gene clusters.

The synthetic production of monodisperse single magnetic domain nanoparticles at ambient temperature is challenging. In nature, magnetosomes--membrane-bound magnetic nanocrystals with unprecedented magnetic properties--can be biomineralized by magnetotactic bacteria. However, these microbes are difficult to handle.

The FtsZ-like protein FtsZm of Magnetospirillum gryphiswaldense likely interacts with its generic homolog and is required for biomineralization under nitrate deprivation.

Midcell selection, septum formation, and cytokinesis in most bacteria are orchestrated by the eukaryotic tubulin homolog FtsZ. The alphaproteobacterium Magnetospirillum gryphiswaldense (MSR-1) septates asymmetrically, and cytokinesis is linked to splitting and segregation of an intracellular chain of membrane-enveloped magnetite crystals (magnetosomes). In addition to a generic, full-length ftsZ gene, MSR-1 contains a truncated ftsZ homolog (ftsZm) which is located adjacent to genes controlling biomineralization and magnetosome chain formation.

The magnetosome proteins MamX, MamZ and MamH are involved in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense.

Magnetospirillum gryphiswaldense uses intracellular chains of membrane-enveloped magnetite crystals, the magnetosomes, to navigate within magnetic fields. The biomineralization of magnetite nanocrystals requires several magnetosome-associated proteins, whose precise functions so far have remained mostly unknown. Here, we analysed the functions of MamX and the Major Facilitator Superfamily (MFS) proteins MamZ and MamH.