Time-resolved cryo-EM (TRCEM) sample preparation using a PDMS-based microfluidic chip assembly.

Unlabelled: Time-resolved cryo-EM (TRCEM) makes it possible to provide structural and kinetic information on a reaction of biomolecules before the equilibrium is reached. Several TRCEM methods have been developed in the past to obtain key insights into the mechanism of action of molecules and molecular machines on the time scale of tens to hundreds of milliseconds, which is unattainable by the normal blotting method. Here we present our TRCEM setup utilizing a polydimethylsiloxane (PDMS)-based microfluidics chip assembly, comprising three components: a PDMS-based, internally SiO -coated micromixer, a glass-capillary microreactor, and a PDMS-based microsprayer for depositing the reaction product onto the EM grid.

Passage through micro-sprayer increases functional activity - implications for activity assays in time-resolved cryo-EM.

This study examines the validity of an assay that is used to report on the retainment of functional competence by ribosomes as they pass a micro-sprayer. We find a reproducible , rather than the expected in GFP production as monitored by fluorescence, which suggests heterogeneity or partial aggregation of ribosomes in solution. An even larger increase in functional activity is observed when sonication is used, pointing to mechanical agitation as the decisive factor in both scenarios.

Inconsistencies in the published rabbit ribosomal rRNAs: a proposal for uniformity in sequence and site numbering.

Examination of all publicly available Oryctolagus cuniculus (rabbit) ribosome cryo-EM structures reveals numerous confusing inconsistencies. First, there are a plethora of single nucleotide differences among the various rabbit 28S and 18S rRNA structures. Second, two nucleotides are absent from the NCBI Reference Sequence for the 18S rRNA gene.

A case of ileal caecum intussusception mimicking rectal prolapse in a 4-month-old female infant.

Introduction And Importance: Ilea caecum Intussusception protruding to the level of anus is a rare manifestation and potentially serious condition in infants.

Case Presentation: A four-month-old infant presented with a one-day history of non-projectile vomiting, three episodes, food contents, worsened by feeding, accompanied by intermittent low-grade fever, and one instance of passing black tarry stool. After outpatient treatment, the infant showed improvement for three days, but later the mother noticed a protruding, self-reducing anal mass, hence the suspected rectal prolapse, which was then Referred for further management.

Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency.

How Dedicated Ribosomes Translate a Leaderless mRNA.

In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in rpsB mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing.

Structural insight into translation initiation of the cl leaderless mRNA.

In bacteriophage λ lysogens, the λcI repressor is encoded by the leaderless transcript (lmRNA) initiated at the λpRM promoter. Translation is enhanced in mutants deficient in ribosomal protein uS2. Although translation initiation of lmRNA is conserved in bacteria, archaea, and eukaryotes, structural insight of a lmRNA translation initiation complex is missing.

Beyond ManifoldEM: geometric relationships between manifold embeddings of a continuum of 3D molecular structures and their 2D projections.

ManifoldEM is an established method of geometric machine learning developed to extract information on conformational motions of molecules from their projections obtained by cryogenic electron microscopy (cryo-EM). In a previous work, in-depth analysis of the properties of manifolds obtained for simulated ground-truth data from molecules exhibiting domain motions has led to improvements of this method, as demonstrated in selected applications of single-particle cryo-EM. In the present work this analysis has been extended to investigate the properties of manifolds constructed by embedding data from synthetic models represented by atomic coordinates in motion, or three-dimensional density maps from biophysical experiments other than single-particle cryo-EM, with extensions to cryo-electron tomography and single-particle imaging with a X-ray free-electron laser.

Time resolution in cryo-EM using a novel PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here we introduce a novel time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency.

Recovery of Conformational Continuum From Single-Particle Cryo-EM Images: Optimization of ManifoldEM Informed by Ground Truth.

This work is based on the manifold-embedding approach to study biological molecules exhibiting continuous conformational changes. Previous work established a method-now termed ManifoldEM-capable of reconstructing 3D movies and accompanying free-energy landscapes from single-particle cryo-EM images of macromolecules exercising multiple conformational degrees of freedom. While ManifoldEM has proven its viability in several experimental studies, critical limitations and uncertainties have been found throughout its extended development and use.

Characterization of N-terminal RYR2 variants outside CPVT1 hotspot regions using patient iPSCs reveal pathogenesis and therapeutic potential.

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is a cardiac channelopathy causing ventricular tachycardia following adrenergic stimulation. Pathogenic variants in RYR2-encoded ryanodine receptor 2 (RYR2) cause CPVT1 and cluster into domains I-IV, with the most N-terminal domain involving residues 77-466. Patient-specific induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated for RYR2-F13L, -L14P, -R15P, and -R176Q variants.

Molecular architecture of 40S translation initiation complexes on the hepatitis C virus IRES.

Hepatitis C virus mRNA contains an internal ribosome entry site (IRES) that mediates end-independent translation initiation, requiring a subset of eukaryotic initiation factors (eIFs). Biochemical studies revealed that direct binding of the IRES to the 40S ribosomal subunit places the initiation codon into the P site, where it base pairs with eIF2-bound Met-tRNAiMet forming a 48S initiation complex. Subsequently, eIF5 and eIF5B mediate subunit joining, yielding an elongation-competent 80S ribosome.

RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis.

Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2.

Symmetric activation and modulation of the human calcium-sensing receptor.

The human extracellular calcium-sensing (CaS) receptor controls plasma Ca levels and contributes to nutrient-dependent maintenance and metabolism of diverse organs. Allosteric modulation of the CaS receptor corrects disorders of calcium homeostasis. Here, we report the cryogenic-electron microscopy reconstructions of a near-full-length CaS receptor in the absence and presence of allosteric modulators.

Contributions of single-particle cryoelectron microscopy toward fighting COVID-19.

Single-particle cryoelectron microscopy (cryo-EM), whose full capabilities have been realized only within the past decade, has had a pivotal role in the fight against COVID-19. This is due to the technique's intrinsic power to depict both structural and dynamic features of molecules; in this case, of the spike protein of SARS-CoV-2. By now, numerous cryo-EM studies have furthered our understanding of spike protein-angiotensin-converting enzyme 2 (ACE2) receptor interactions, which has informed the design of effective vaccines, and have enabled the characterization of neutralizing antibody binding sites, which will lead to the design of novel therapeutics as the virus evolves.

What is in the black box? - A perspective on software in cryoelectron microscopy.

This article bemoans the demise of truly modular open-source image processing systems, such as SPIDER, in recent years' development of tools for three-dimensional reconstruction in cryo-electron microscopy. Instead, today's users have to rely on the functionality of software systems that have little or no transparency. As a consequence, users of such packages no longer gain a conceptual understanding and intuitive grasp of the analytical routes leading from the stream of input data to the final density map.

A glycan gate controls opening of the SARS-CoV-2 spike protein.

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded 'down' to an exposed 'up' state to bind the human angiotensin-converting enzyme 2 receptor and infect cells. While snapshots of the 'up' and 'down' states have been obtained by cryo-electron microscopy and cryo-electron tomagraphy, details of the RBD-opening transition evade experimental characterization. Here over 130 µs of weighted ensemble simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD-opening pathways.

"Just in Time": The Role of Cryo-Electron Microscopy in Combating Recent Pandemics.

Single-particle cryogenic electron microscopy (cryo-EM), whose full power was not realized until the advent of powerful detectors in 2012, has a unique position as a method of structure determination as it is capable of providing information about not only the structure but also the dynamical features of biomolecules. This information is of special importance in understanding virus-host interaction and explains the crucial role of cryo-EM in the efforts to find vaccinations and cures for pandemics the world has experienced in the past decade.

The Processing of Visual Signals in Major Depressive Disorder.

The balanced processing of the internal mental world and the external world is a crucial aspect of everyday well-being. An extensive control of the internal emotional and cognitive world that often results in an internal expression of distress is a common feature of internalizing disorders. However, how depression affects the processing of the external world is still an open question.

[Personality organization and mentalization of depressive inpatients in a long-term-study].

In a naturalistic long-term follow-up design this study investigated the improvement of depressive symptom severity, mentalization deficiency and personality organization. 300 patients with depressive symptoms were assessed at three evaluation times (before therapy, after therapy and one to three years after discharge) with the Patient Health Questionnaire Depression Scale (PHQ-9), the Mentalization Questionnaire (MZQ) and the Inventory of Personality Organization (IPO-16). Patients improved significantly in depressive symptom severity with strong impact.

A glycan gate controls opening of the SARS-CoV-2 spike protein.

SARS-CoV-2 infection is controlled by the opening of the spike protein receptor binding domain (RBD), which transitions from a glycan-shielded "down" to an exposed "up" state in order to bind the human ACE2 receptor and infect cells. While snapshots of the "up" and "down" states have been obtained by cryoEM and cryoET, details of the RBD opening transition evade experimental characterization. Here, over 130 μs of weighted ensemble (WE) simulations of the fully glycosylated spike ectodomain allow us to characterize more than 300 continuous, kinetically unbiased RBD opening pathways.

Interaction Networks of Ribosomal Expansion Segments in Kinetoplastids.

Expansion segments (ES) are insertions of a few to hundreds of nucleotides at discrete locations on eukaryotic ribosomal RNA (rRNA) chains. Some cluster around 'hot spots' involved in translation regulation and some may participate in biogenesis. Whether ES play the same roles in different organisms is currently unclear, especially since their size may vary dramatically from one species to another and very little is known about their functions.

Nanobubble-controlled nanofluidic transport.

Nanofluidic platforms offering tunable material transport are applicable in biosensing, chemical detection, and filtration. Prior studies have achieved selective and controllable ion transport through electrical, optical, or chemical gating of complex nanostructures. Here, we mechanically control nanofluidic transport using nanobubbles.

A Time-Resolved Cryo-EM Study of Saccharomyces cerevisiae 80S Ribosome Protein Composition in Response to a Change in Carbon Source.

The role of the ribosome in the regulation of gene expression has come into increased focus. It is proposed that ribosomes are catalytic engines capable of changing their protein composition in response to environmental stimuli. Time-resolved cryo-electron microscopy (cryo-EM) techniques are employed to identify quantitative changes in the protein composition and structure of the Saccharomyces cerevisiae 80S ribosomes after shifting the carbon source from glucose to glycerol.