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. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140 ms.

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. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. By employing this method, we show for the first time the mechanism of progressive HlfX-mediated splitting of the 70S E. coli ribosome in the presence of the GTP, via capture of three high-resolution reaction intermediates within 140 ms.

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. Initiation can also proceed without eIF2, in which case Met-tRNAiMet is recruited directly by eIF5B. However, the structures of initiation complexes assembled on the HCV IRES, the transitions between different states, and the accompanying conformational changes have remained unknown. To fill these gaps, we now obtained cryo-EM structures of IRES initiation complexes, at resolutions up to 3.5 Å, that cover all major stages from the initial ribosomal association, through eIF2-containing 48S initiation complexes, to eIF5B-containing complexes immediately prior to subunit joining. These structures provide insights into the dynamic network of 40S/IRES contacts, highlight the role of IRES domain II, and reveal conformational changes that occur during the transition from eIF2- to eIF5B-containing 48S complexes and prepare them for subunit joining.

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. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.

Advances in domain and subunit localization technology for electron microscopy.

The award of the 2017 Nobel Prize in chemistry, 'for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution', was recognition that this method, and electron microscopy more generally, represent powerful techniques in the scientific armamentarium for atomic level structural assessment. Technical advances in equipment, software, and sample preparation, have allowed for high-resolution structural determination of a range of complex biological machinery such that the position of individual atoms within these mega-structures can be determined. However, not all targets are amenable to attaining such high-resolution structures and some may only be resolved at so-called intermediate resolutions. In these cases, other tools are needed to correctly characterize the domain or subunit orientation and architecture. In this review, we will outline various methods that can provide additional information to help understand the macro-level organization of proteins/biomolecular complexes when high-resolution structural description is not available. In particular, we will discuss the recent development and use of a novel protein purification approach, known as the the PA tag/NZ-1 antibody system, which provides numberous beneficial properties, when used in electron microscopy experimentation.

Attraction of posture and motion-trajectory elements of conspecific biological motion in medaka fish.

Visual recognition of conspecifics is necessary for a wide range of social behaviours in many animals. Medaka (Japanese rice fish), a commonly used model organism, are known to be attracted by the biological motion of conspecifics. However, biological motion is a composite of both body-shape motion and entire-field motion trajectory (i.e., posture or motion-trajectory elements, respectively), and it has not been revealed which element mediates the attractiveness. Here, we show that either posture or motion-trajectory elements alone can attract medaka. We decomposed biological motion of the medaka into the two elements and synthesized visual stimuli that contain both, either, or none of the two elements. We found that medaka were attracted by visual stimuli that contain at least one of the two elements. In the context of other known static visual information regarding the medaka, the potential multiplicity of information regarding conspecific recognition has further accumulated. Our strategy of decomposing biological motion into these partial elements is applicable to other animals, and further studies using this technique will enhance the basic understanding of visual recognition of conspecifics.

The PA Tag: A Versatile Peptide Tagging System in the Era of Integrative Structural Biology.

We have recently developed a novel protein tagging system based on the high affinity interaction between an antibody NZ-1 and its antigen PA peptide, a dodecapeptide that forms a ß-turn in the binding pocket of NZ-1. This unique conformation allows for the PA peptide to be inserted into turn-forming loops within a folded protein domain and the system has been variously used in general applications including protein purification, Western blotting and flow cytometry, or in more specialized applications such as reporting protein conformational change, and identifying subunits of macromolecular complexes with electron microscopy. Thus the small and "portable" nature of the PA tag system offers a versatile and powerful tool that can be implemented in various aspects of integrative structural biology.

Development of a new protein labeling system to map subunits and domains of macromolecular complexes for electron microscopy.

Several gene fusion technologies have been successfully applied to label particular subunits or domains within macromolecular complexes to enable positional mapping of electron microscopy (EM) density maps, but exogenous fusion of a protein domain into the target polypeptide can cause unwanted structural and functional outcomes. Fab fragments from antibodies can be used as labeling reagents during EM visualization without gene manipulation of the target protein, but this method requires a panel of high-affinity antibodies that recognize a wide variety of epitopes. Linear peptide tags and their anti-tag antibodies can be used but they have a limited mapping ability as their placement is usually limited to the terminal regions of a protein. The PA dodecapeptide epitope tag (GVAMPGAEDDVV), forms a tight ß-turn in the antigen binding pocket of its antibody (NZ-1). This capability allows for insertion of the PA tag into various surface-exposed loops within a multi-domain cell adhesion receptor, αIIbß3 integrin. We confirmed that the purified PA-tagged integrin ectodomain fragments can form a stable complex with NZ-1 Fab. Negative stain EM of the various integrin-NZ-1 complexes revealed that a majority of the particles exhibited a clear density corresponding to the NZ-1 Fab; and the positions of the bound Fab were in good agreement with the predicted location of the inserted PA tag. The high-affinity and insertion-compatibility of the PA tag system allowed us to develop a new EM labeling methodology applicable to proteins for which good antibodies are not available.

Protein-induced mass increase of the gastrointestinal tract of locusts improves net nutrient uptake via larger meals rather than more efficient nutrient absorption.

Increasing the tissue biomass and/or volume of the gastrointestinal tract (GIT) is commonly seen when animals feed on poor-quality diets. This increase can simply permit larger meal sizes, but may also rebalance nutritionally imbalanced ingesta by allowing selective absorption of limiting nutrients. In an insect herbivore, the migratory locust, a synthetic diet with a high ratio of protein to carbohydrate was found to induce mass enhancement of the GIT. When normalised for sex and overall body size, increases to the mass of the foregut and midgut caeca resulted in higher absorption (20-30%) of both protein and carbohydrate when subsequently feeding on three chemically and structurally different grasses. Greater net absorption of macronutrients occurred because these locusts ate larger meals that transited at the same time and with the same digestive efficiency as locusts in which the GIT was not enlarged. Thus, plasticity of the GIT did not improve nutritional homeostasis, but increased the rate of nutrient uptake.