Abstract Visual proteomics attempts to build atlases of the molecular content cells but automated annotation cryo electron tomograms remains challenging. Template matching (TM) and methods based on machine learning detect structural signatures macromolecules. However, their applicability limited in terms both abundance size targets. Here we show that performance TM is greatly improved by using template-specific search parameter optimization including higher-resolution information. We...

Abstract Sample fixation by vitrification is critical for the optimal structural preservation of biomolecules and subsequent high-resolution imaging cryo-correlative light electron microscopy (cryoCLEM). There a large resolution gap between cryo fluorescence (cryoFLM), ~400-nm, sub-nanometre achievable with cryo-electron (cryoEM), which hinders interpretation cryoCLEM data. Here, we present general approach to increase cryoFLM using cryo-super-resolution (cryoSR) that compatible successive...

Ribosomes translate genetic information into primary structure. During translation, various cofactors transiently bind to the ribosome that undergoes prominent conformational and structural changes. Different translational states of ribosomes have been well characterized in vitro. However, which extent known are representative native situation inside cells has thus far only addressed prokaryotes. Here, we apply cryo-electron tomography cryo-FIB milled Dictyostelium discoideum combined with...

Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and structurally analyze biomolecules in situ by subtomogram averaging, yet data quality critically depends on specimen thickness. Cells that are too thick for transmission imaging can be thinned into lamellae cryo-focused ion beam (cryo-FIB) milling. Despite being crucial parameter directly affecting attainable resolution, optimal lamella thickness has not been systematically investigated nor the...

Solid-state nanopores are an emerging class of single-molecule sensors. Whereas most studies so far focused on double-stranded DNA (dsDNA) molecules, exploration single-stranded (ssDNA) is great interest as well, for example to employ such a nanopore device read out the sequence. Here, we study translocation long random-sequence ssDNA through nanopores. Using atomic force microscopy, observe hybridize into random coil, forming blobs around 100 nm in diameter 7 kb ssDNA. These large entangled...

Ribosome assembly in eukaryotes involves the activity of hundreds factors that direct hierarchical ribosomal proteins and numerous RNA folding steps. However, detailed insights into function events are lacking. To address this, we have developed ChemModSeq, a method combines structure probing, high-throughput sequencing statistical modeling, to quantitatively measure structural rearrangements during macromolecular complexes. By applying ChemModSeq purified 40S intermediates obtained...

Cryogenic transmission electron microscopy (cryo-TEM) and super-resolution fluorescence are two popular ever improving methods for high-resolution imaging of biological samples. In recent years, the combination these techniques into one correlated workflow has gained attention as a promising route towards contextualizing enriching cryo-TEM imagery. A problem that is often encountered in light-induced damage to sample during renders structure unsuitable TEM imaging. this paper, we describe...

Abstract Cryo-electron tomography (CryoET) resolves individual macromolecules inside living cells. However, the complex composition and high density of cells challenge faithful identification features in tomograms. Here, we capitalize on recent advances electron demonstrate that 3D template matching (TM) localizes a wide range structures crowded eukaryotic with confidence 10 to 100-fold above noise level. We establish TM pipeline systematically tuned parameters for automated, objective...

We investigate translocation of linear and circular double-stranded DNA molecules through solid state nanopores where each molecule is recaptured re-translocated many times. Single can be by switching voltage polarity for hundreds or even thousands The large number recapture events allows statistics on the individual molecules. Surprisingly, we observe that do not translocate in a head-to-tail fashion, but instead as folded blob multiple parts simultaneously pore parallel. This folding...

Pathogens frequently produce proteins to evade or inhibit host immune responses. One such protein is lymphostatin from attaching and effacing Escherichia coli (also known as lymphocyte inhibitory factor A; LifA), which influences intestinal colonization inhibits mitogen- antigen-activated proliferation of T lymphocytes pro-inflammatory cytokine synthesis. Here, we report the cloning, purification characterization a LifA homologue Chlamydia pecorum. The predicted 382 KDa (CPE2_0552) exhibited...

Abstract Eukaryotes have a multitude of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, from kinetoplastids divergent. The structural functional consequences this variation unknown. Here, we biochemically structurally characterised nucleosome core particles (NCPs) kinetoplastid parasite Trypanosoma brucei. A structure T. brucei NCP reveals global histone architecture is conserved, specific sequence...

Microtubule (MT) nucleation depends on the γ-tubulin complex (γ-TuC), in which multiple copies of heterotetrameric small (γ-TuSC) associate to form a ring-like structure (in metazoans, ring complex; γ-TuRC) [1-7]. Additional conserved regulators γ-TuC include protein Mzt1 (MOZART1 human; GIP1/1B and GIP2/1A plants) [8-13] proteins containing Centrosomin Motif 1 (CM1) domain [10, 14-19]. Many insights into have come from vivo analysis fission yeast Schizosaccharomyces pombe. The S. pombe CM1...

Abstract Cryo-electron tomography (cryo-ET) is a powerful method to elucidate subcellular architecture and structurally analyse biomolecules in situ by subtomogram averaging (STA). Specimen thickness key factor affecting cryo-ET data quality. Cells that are too thick for transmission imaging can be thinned cryo-focused-ion-beam (cryo-FIB) milling. However, optimal specimen on lamellae has not been systematically investigated. Furthermore, the ions used ablate material cause damage lamellae,...

Abstract Correlative light-electron microscopy (CLEM) combines the high spatial resolution of transmission electron (TEM) with capability fluorescence light (FLM) to locate rare or transient cellular events within a large field view. CLEM is therefore powerful technique study processes. Aligning images derived from both imaging modalities prerequisite correlate two data sets, and poor alignment can limit interpretability data. Here, we describe how uranyl acetate, commonly-used contrast...

Harnessing bacterial processes for the selective separation of strategic and critical metals contained in spent lithium-ion batteries (LIBs).

Abstract Super‐resolution light microscopy (SRM) enables imaging of biomolecules within cells with nanometer precision. Cryo‐fixation by vitrification offers optimal structure preservation biological specimens and permits sequential cryo electron (cryoEM) on the same sample, but is rarely used for SRM due to various technical challenges lack fluorophores developed vitrified conditions. Here, a protocol perform correlated cryoSRM cryoEM intact mammalian using fluorescent proteins commercially...

Abstract Eukaryotes have a multitude of diverse mechanisms for organising and using their genomes, but the histones that make up chromatin are highly conserved. Unusually, from kinetoplastids divergent. The structural functional consequences this variation unknown. Here, we biochemically structurally characterised nucleosome core particles (NCPs) kinetoplastid parasite Trypanosoma brucei . A structure T. NCP reveals global histone architecture is conserved, specific sequence alterations lead...

Cryo-electron tomography enables the visualization of macromolecular complexes within native cellular environments, but is limited by incomplete angular sampling and maximal electron dose that biological specimen can be exposed to. Here, we developed cryoTIGER (Tilt Interpolation Generator for Enhanced Reconstruction), a computational workflow leveraging deep learning-based frame interpolation to generate intermediate tilt images. By interpolating between series projections, improves...

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