In situ structural insights into the excitation-contraction coupling mechanism of skeletal muscle.

Excitation-contraction coupling (ECC) is a fundamental mechanism in control of skeletal muscle contraction and occurs at triad junctions, where dihydropyridine receptors (DHPRs) on transverse tubules sense excitation signals and then cause calcium release from the sarcoplasmic reticulum via coupling to type 1 ryanodine receptors (RyR1s), inducing the subsequent contraction of muscle filaments. However, the molecular mechanism remains unclear due to the lack of structural details. Here, we explored the architecture of triad junction by cryo-electron tomography, solved the in situ structure of RyR1 in complex with FKBP12 and calmodulin with the resolution of 16.7 Angstrom, and found the intact RyR1-DHPR supercomplex. RyR1s arrange into two rows on the terminal cisternae membrane by forming right-hand corner-to-corner contacts, and tetrads of DHPRs bind to RyR1s in an alternating manner, forming another two rows on the transverse tubule membrane. This unique arrangement is important for synergistic calcium release and provides direct evidence of physical coupling in ECC.

Author Correction: Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

A Correction to this paper has been published: https://doi.org/10.1038/s41551-021-00725-w.

Reprint of "Amorphous nickel titanium alloy film: A new choice for cryo electron microscopy sample preparation".

Cryo-electron microscopy (cryoEM) has become one of the most important approach for structural biology. However, barriers are still there for an increased successful rate, a better resolution and improved efficiency from sample preparation, data collection to image processing. CryoEM sample preparation is one of the bottlenecks with many efforts made recently, including the optimization of supporting substrate (e.g. ultra-thin carbon, graphene, pure gold, 2d crystal of streptavidin, and affinity modification), which was aimed to solve air-water interface problem, or reduce beam induced motion (BIM), or change particle distribution in the grid hole. Here, we report another effort of developing a new supporting substrate, the amorphous nickel-titanium alloy (ANTA) film, for cryoEM sample preparation as a layer of holey supporting film covering on TEM grid. Our investigations showed advantages of ANTA film in comparison with conventional carbon film, including much better electron conductivity and trace non-specific interaction with protein. These advantages yield less BIM and significantly improved particle distribution during cryoEM experiment of human apo-ferritn, thus resulting an improved reconstruction resolution from a reduced number of micrographs and particles. Unlike the pure gold film, the usage of the ANTA film is just same with the carbon film, compatible to conventional automatic cryoEM data collection procedure.

AgsA oligomer acts as a functional unit.

AgsA (aggregation-suppressing protein) is an ATP-independent molecular chaperone machine belonging to the family of small heat shock proteins (sHSP), and it can prevent the aggregation of non-natural proteins. However, the substrate-binding site of AgsA and the functional unit that captures and binds the substrate remain unknown. In this study, different N-terminal and C-terminal deletion mutants of AgsA were constructed and their effects on AgsA oligomer assembly and chaperone activity were investigated. We found that the IXI motif at the C-terminus and the α-helix at the N-terminus affected the oligomerization and molecular chaperone activity of AgsA. In this work, we obtained a 6.8 Å resolution structure of AgsA using Electron cryo-microscopy (cryo-EM), and found that the functional form of AgsA was an 18-mer with D3 symmetry. Through amino acid mutations, disulfide bonds were introduced into two oligomeric interfaces, namely dimeric interface and non-partner interface. Under oxidation and reduction conditions, the chaperone activity of the disulfide-bonded AgsA did not change significantly, indicating that AgsA would not dissociate to achieve chaperone activity. Therefore, we concluded that the oligomer, especially 18-mer, was the primary functional unit.

Amorphous nickel titanium alloy film: A new choice for cryo electron microscopy sample preparation.

Cryo-electron microscopy (cryoEM) has become one of the most important approach for structural biology. However, barriers are still there for an increased successful rate, a better resolution and improved efficiency from sample preparation, data collection to image processing. CryoEM sample preparation is one of the bottlenecks with many efforts made recently, including the optimization of supporting substrate (e.g. ultra-thin carbon, graphene, pure gold, 2d crystal of streptavidin, and affinity modification), which was aimed to solve air-water interface problem, or reduce beam induced motion (BIM), or change particle distribution in the grid hole. Here, we report another effort of developing a new supporting substrate, the amorphous nickel-titanium alloy (ANTA) film, for cryoEM sample preparation as a layer of holey supporting film covering on TEM grid. Our investigations showed advantages of ANTA film in comparison with conventional carbon film, including much better electron conductivity and trace non-specific interaction with protein. These advantages yield less BIM and significantly improved particle distribution during cryoEM experiment of human apo-ferritn, thus resulting an improved reconstruction resolution from a reduced number of micrographs and particles. Unlike the pure gold film, the usage of the ANTA film is just same with the carbon film, compatible to conventional automatic cryoEM data collection procedure.

Large-scale generation of functional mRNA-encapsulating exosomes via cellular nanoporation.

Exosomes are attractive as nucleic-acid carriers because of their favourable pharmacokinetic and immunological properties and their ability to penetrate physiological barriers that are impermeable to synthetic drug-delivery vehicles. However, inserting exogenous nucleic acids, especially large messenger RNAs, into cell-secreted exosomes leads to low yields. Here we report a cellular-nanoporation method for the production of large quantities of exosomes containing therapeutic mRNAs and targeting peptides. We transfected various source cells with plasmid DNAs and stimulated the cells with a focal and transient electrical stimulus that promotes the release of exosomes carrying transcribed mRNAs and targeting peptides. Compared with bulk electroporation and other exosome-production strategies, cellular nanoporation produced up to 50-fold more exosomes and a more than 103-fold increase in exosomal mRNA transcripts, even from cells with low basal levels of exosome secretion. In orthotopic phosphatase and tensin homologue (PTEN)-deficient glioma mouse models, mRNA-containing exosomes restored tumour-suppressor function, enhanced inhibition of tumour growth and increased survival. Cellular nanoporation may enable the use of exosomes as a universal nucleic-acid carrier for applications requiring transcriptional manipulation.

Characterization of influenza virus PR8 strain cultured in embryonated eggs by cryo-electron tomography.

Influenza A viruses, as causative agents of seasonal epidemics and periodic worldwide pandemics, cause enormous mortality loss globally. The PR8 strain cultured in chicken eggs is widely used for scientific research and the production of influenza vaccines. Here, based on Cryo-electron Tomography (CET), we analyzed the morphological and structural characteristics of the influenza virus PR8 strain at different pHs. We found that a large number of defective virions were propagated in embryonated eggs. By comparing virions with/without the matrix layer, it was revealed that the matrix layer played an essential role in the structural integrity of virions and RNPs encapsulation during the influenza virus life cycle. We also utilized hemagglutinin receptor-containing liposomes to mimic the membrane fusion process. Several potential intermediates of HA during membrane fusion were observed at acidic pH. Our observations afford insight into the architecture and function of influenza virus.

Interaction of the Homer1 EVH1 domain and skeletal muscle ryanodine receptor.

The skeletal muscle ryanodine receptor (RyR1) proteins are intracellular calcium (Ca2+) release channels on the membrane of the sarcoplasmic reticulum (SR) and required for skeletal muscle excitation-contraction coupling. Homer (Vesl) is a family of scaffolding proteins that modulate target proteins including RyRs (ryanodine receptors), mGluRs (group 1 metabotropic glutamate receptors) and IP3Rs (inositol-1,4,5-trisphosphate receptors) through a conserved EVH1 (Ena/VASP homology 1) domain. Here, we examined the interaction between Homer1 EVH1 domain and RyR1 by co-immunoprecipitation, continuous sucrose density-gradient centrifugation, and bio-layer interferometry binding assay at different Ca2+ concentrations. Our results show that there exists a high-affinity binding between the Homer1 EVH1 domain and RyR1, especially at 1 mM of Ca2+. Based on our data and the known structures of Homer1 EVH1 domain and RyR1, we found two consensus proline-rich sequences in the structure of RyR1, PPHHF and FLPPP, and proposed two corresponding binding models to show mechanisms of recognition different from those used by other proline-rich motifs. The side proline residues of two proline-rich motifs from RyR1 are away from the hydrophobic surface of Homer1 EVH1, rather than buried in this hydrophobic surface. Our results provide evidence that Homer1 regulates RyR1 by direct interaction.

The insecticide chlorantraniliprole is a weak activator of mammalian skeletal ryanodine receptor/Ca2+ release channel.

Chlorantraniliprobe (Chlo), a potent insecticide, demolishes intracellular Ca2+ homeostasis of insects by inducing uncontrolled Ca2+ release through ryanodine receptors (RyRs). Chlo is lethal to insects but has low toxicity to mammals. In this study, we investigated the effects of Chlo on RyR1 from mammalian skeletal muscle. Ca2+ release assay indicated that Chlo at high concentrations promoted Ca2+ release from sarcoplasmic reticulum through RyR1 channels. Single channel recording of purified RyR1 showed that Chlo activated RyR1 channel, increased channel open probability Po, reduced channel mean close time Tc, but did not change the channel mean open time To, suggesting that Chlo destabilized the closed RyR1 channel, rendered the channel easy to open. The dissociation constant Kd values of Chlo for RyR1 were of micromolar level, approximately 100-fold larger than that for insect RyR. The Kd values were smaller for open states than for closed/blocked states of the RyR1 channel. The maximal binding capacity Bmax did not change in the presence of either channel activators or inhibitors/blockers. Our results demonstrate that the insecticide Chlo is a weak activator of mammalian RyR1. It can interact with mammalian RyR1 and activate RyR1 channel but with much lower affinity compared with insect RyR; Chlo has a binding site distinct from all known RyR channel modulators and represents a novel type of RyR channel modulator. Our data provide biochemical and pharmacological insights into its high specificity to insect RyR and high selectivity of poisoning to insects over mammals.

Structural and functional characterization of polyethylene terephthalate hydrolase from Ideonella sakaiensis.

Polyethylene terephthalate (PET) hydrolase from Ideonella sakaiensis (IsPETase) can be used to degrade PET. In order to use IsPETase in industry, we studied the enzymatic activity of IsPETase in different conditions containing environmental and physicochemical factors commonly found in nature. We observed that salts and glycerol enhanced the enzymatic activity, while detergents and organic solvents reduced the enzymatic activity. IsPETase hydrolyzed p-nitrophenyl (p-NP) esters instead of naphthyl esters. To make IsPETase an enzyme capable of hydrolyzing naphthyl esters, site-directed mutagenesis was carried out based on the structural information provided by the crystal structure. We found that the IsPETaseS93M, IsPETaseW159F, and IsPETaseN241F mutants can hydrolyze naphthyl esters. IsPETase engineering can direct researchers to use this α/ß-hydrolase protein scaffold to design enzymes that can hydrolyze a variety of polyesters.

Increased cellular uptake of peptide-modified PEGylated gold nanoparticles.

Gold nanoparticles are promising drug delivery vehicles for nucleic acids, small molecules, and proteins, allowing various modifications on the particle surface. However, the instability and low bioavailability of gold nanoparticles compromise their clinical application. Here, we functionalized gold nanoparticles with CPP fragments (CALNNPFVYLI, CALRRRRRRRR) through sulfhydryl PEG to increase their stability and bioavailability. The resulting gold nanoparticles were characterized with transmission electron microscopy (TEM), dynamic light scattering (DLS), UV-visible spectrometry and X-ray photoelectron spectroscopy (XPS), and the stability in biological solutions was evaluated. Comparing to PEGylated gold nanoparticles, CPP (CALNNPFVYLI, CALRRRRRRRR)-modified gold nanoparticles showed 46 folds increase in cellular uptake in A549 and B16 cell lines, as evidenced by the inductively coupled plasma atomic emission spectroscopy (ICP-AES). The interactions between gold nanoparticles and liposomes indicated CPP-modified gold nanoparticles bind to cell membrane more effectively than PEGylated gold nanoparticles. Surface plasmon resonance (SPR) was used to measure interactions between nanoparticles and the membrane. TEM and uptake inhibitor experiments indicated that the cellular entry of gold nanoparticles was mediated by clathrin and macropinocytosis. Other energy independent endocytosis pathways were also identified. Our work revealed a new strategy to modify gold nanoparticles with CPP and illustrated the cellular uptake pathway of CPP-modified gold nanoparticles.

iTRAQ protein profile analysis of developmental dynamics in soybean [Glycine max (L.) Merr.] leaves.

Zao5241 is an elite soybean [Glycine max (L.) Merr.] line and backbone parent. In this study, we employed iTRAQ to analyze the proteomes and protein expression profiles of Zao5241 during leaf development. We identified 1,245 proteins in all experiments, of which only 45 had been previously annotated. Among overlapping proteins between three biological replicates, 598 proteins with 2 unique peptides identified were reliably quantified. The protein datasets were classified into 36 GO functional terms, and the photosynthesis term was most significantly enriched. A total of 113 proteins were defined as being differentially expressed during leaf development; 41 proteins were found to be differently expressed between two and four week old leaves, and 84 proteins were found to be differently expressed between two and six week old leaves, respectively. Cluster analysis of the data revealed dynamic proteomes. Proteins annotated as electron carrier activity were greatly enriched in the peak expression profiles, and photosynthesis proteins were negatively modulated along the whole time course. This dataset will serve as the foundation for a systems biology approach to understanding photosynthetic development.

Allosteric conformational changes of human HBV core protein transform its assembly.

Hepatitis B Virus core protein (HBc) has multiple roles in the viral lifecycle: viral assembly, compartment for reverse transcription, intracellular trafficking, and nuclear functions. HBc displays assembly polymorphism - it can assemble into icosahedral capsid and aberrant non-capsid structures. It has been hypothesized that the assembly polymorphism is due to allosteric conformational changes of HBc dimer, the smallest assembly unit, however, the mechanism governing the polymorphic assembly of the HBc dimer is still elusive. By using the experimental antiviral drug BAY 41-4109, we successfully transformed the HBc assembly from icosahedral capsid to helical tube. Structural analyses of HBc dimers from helical tubes, T = 4 icosahedral capsid, and sheet-like HBc ensemble revealed differences within the inter-dimer interface. Disruption of the HBc inter-dimer interface may likely promote the various assembly forms of HBc. Our work provides new structural insights into the HBV assembly mechanism and strategic guide for anti-HBV drug design.

Simultaneous microbial reduction of vanadium (V) and chromium (VI) by Shewanella loihica PV-4.

Toxic vanadium (V) and chromium (VI) often co-exist in wastewater from vanadium ore smelting and their reductions by bacterial strain Shewanella loihica PV-4 is realized simultaneously. After 27-d operation, 71.3% of V(V) and 91.2% of Cr(VI) were removed respectively, with citrate as organic carbon source. Enhancement of Cr(VI) bioreduction was observed with the suppressed V(V) reduction. V(IV) and Cr(III), the main reduction products, precipitated inside the organisms and attached on cell surfaces. Both membrane components containing cytochrome c and cytoplasmic fractions containing soluble proteins as well as NADH may contribute to these microbial reductions. Most Cr(VI) were reduced extracellularly and V(V) tended to be reduced through intracellular process, as revealed by mapping the microbial surface and a line scan across the cell, performed by scanning transmission electron microscopy. This study provides an efficient alternative for controlling combined pollution caused by these two metals based on microbial technology.

Unsupervised Cryo-EM Data Clustering through Adaptively Constrained K-Means Algorithm.

In single-particle cryo-electron microscopy (cryo-EM), K-means clustering algorithm is widely used in unsupervised 2D classification of projection images of biological macromolecules. 3D ab initio reconstruction requires accurate unsupervised classification in order to separate molecular projections of distinct orientations. Due to background noise in single-particle images and uncertainty of molecular orientations, traditional K-means clustering algorithm may classify images into wrong classes and produce classes with a large variation in membership. Overcoming these limitations requires further development on clustering algorithms for cryo-EM data analysis. We propose a novel unsupervised data clustering method building upon the traditional K-means algorithm. By introducing an adaptive constraint term in the objective function, our algorithm not only avoids a large variation in class sizes but also produces more accurate data clustering. Applications of this approach to both simulated and experimental cryo-EM data demonstrate that our algorithm is a significantly improved alterative to the traditional K-means algorithm in single-particle cryo-EM analysis.

Structural insights into Ca(2+)-activated long-range allosteric channel gating of RyR1.

Ryanodine receptors (RyRs) are a class of giant ion channels with molecular mass over 2.2 mega-Daltons. These channels mediate calcium signaling in a variety of cells. Since more than 80% of the RyR protein is folded into the cytoplasmic assembly and the remaining residues form the transmembrane domain, it has been hypothesized that the activation and regulation of RyR channels occur through an as yet uncharacterized long-range allosteric mechanism. Here we report the characterization of a Ca(2+)-activated open-state RyR1 structure by cryo-electron microscopy. The structure has an overall resolution of 4.9 Å and a resolution of 4.2 Å for the core region. In comparison with the previously determined apo/closed-state structure, we observed long-range allosteric gating of the channel upon Ca(2+) activation. In-depth structural analyses elucidated a novel channel-gating mechanism and a novel ion selectivity mechanism of RyR1. Our work not only provides structural insights into the molecular mechanisms of channel gating and regulation of RyRs, but also sheds light on structural basis for channel-gating and ion selectivity mechanisms for the six-transmembrane-helix cation channel family.

Peptide Biosynthesis with Stable Isotope Labeling from a Cell-free Expression System for Targeted Proteomics with Absolute Quantification.

Because of its specificity and sensitivity, targeted proteomics using mass spectrometry for multiple reaction monitoring is a powerful tool to detect and quantify pre-selected peptides from a complex background and facilitates the absolute quantification of peptides using isotope-labeled forms as internal standards. How to generate isotope-labeled peptides remains an urgent challenge for accurately quantitative targeted proteomics on a large scale. Herein, we propose that isotope-labeled peptides fused with a quantitative tag could be synthesized through an expression system in vitro, and the homemade peptides could be enriched by magnetic beads with tag-affinity and globally quantified based on the corresponding multiple reaction monitoring signals provided by the fused tag. An Escherichia coli cell-free protein expression system, protein synthesis using recombinant elements, was adopted for the synthesis of isotope-labeled peptides fused with Strep-tag. Through a series of optimizations, we enabled efficient expression of the labeled peptides such that, after Strep-Tactin affinity enrichment, the peptide yield was acceptable in scale for quantification, and the peptides could be completely digested by trypsin to release the Strep-tag for quantification. Moreover, these recombinant peptides could be employed in the same way as synthetic peptides for multiple reaction monitoring applications and are likely more economical and useful in a laboratory for the scale of targeted proteomics. As an application, we synthesized four isotope-labeled glutathione S-transferase (GST) peptides and added them to mouse sera pre-treated with GST affinity resin as internal standards. A quantitative assay of the synthesized GST peptides confirmed the absolute GST quantification in mouse sera to be measurable and reproducible.

iTRAQ-based analysis of developmental dynamics in the soybean leaf proteome reveals pathways associated with leaf photosynthetic rate.

Photosynthetic rate which acts as a vital limiting factor largely affects the potential of soybean production, especially during the senescence phase. However, the physiological and molecular mechanisms that underlying the change of photosynthetic rate during the developmental process of soybean leaves remain unclear. In this study, we compared the protein dynamics during the developmental process of leaves between the soybean cultivar Hobbit and the high-photosynthetic rate cultivar JD 17 using the iTRAQ (isobaric tags for relative and absolute quantification) method. A total number of 1269 proteins were detected in the leaves of these two cultivars at three different developmental stages. These proteins were classified into nine expression patterns depending on the expression levels at different developmental stages, and the proteins in each pattern were also further classified into three large groups and 20 small groups depending on the protein functions. Only 3.05-6.53 % of the detected proteins presented a differential expression pattern between these two cultivars. Enrichment factor analysis indicated that proteins involved in photosynthesis composed an important category. The expressions of photosynthesis-related proteins were also further confirmed by western blotting. Together, our results suggested that the reduction in photosynthetic rate as well as chloroplast activity and composition during the developmental process was a highly regulated and complex process which involved a serial of proteins that function as potential candidates to be targeted by biotechnological approaches for the improvement of photosynthetic rate and production.

A local-optimization refinement algorithm in single particle analysis for macromolecular complex with multiple rigid modules.

Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macromolecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate alignments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orientations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimensional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large (60S/50S) and the small (40S/30S) subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolution single particle analysis of macromolecular complexes with dynamic conformations.