Molecular Mechanism of pH-Induced Protrusion Configuration Switching in Piscine Betanodavirus Implies a Novel Antiviral Strategy.

Many viruses contain surface spikes or protrusions that are essential for virus entry. These surface structures can thereby be targeted by antiviral drugs to treat viral infections. Nervous necrosis virus (NNV), a simple nonenveloped virus in the genus of betanodavirus, infects fish and damages aquaculture worldwide.

Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å.

While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis.

Site-directed biochemical analyses reveal that the switchable C-terminus of Rpc31 contributes to RNA polymerase III transcription initiation.

Rpc31 is a subunit in the TFIIE-related Rpc82/34/31 heterotrimeric subcomplex of Saccharomyces cerevisiae RNA polymerase III (pol III). Structural analyses of pol III have indicated that the N-terminal region of Rpc31 anchors on Rpc82 and further interacts with the polymerase core and stalk subcomplex. However, structural and functional information for the C-terminal region of Rpc31 is sparse.

Cryo-EM Analyses Permit Visualization of Structural Polymorphism of Biological Macromolecules.

The functions of biological macromolecules are often associated with conformational malleability of the structures. This phenomenon of chemically identical molecules with different structures is coined structural polymorphism. Conventionally, structural polymorphism is observed directly by structural determination at the density map level from X-ray crystal diffraction.

Catalytic machinery of methane oxidation in particulate methane monooxygenase (pMMO).

In this focused review, we portray the recently reported 2.5 Å cyro-EM structure of the particulate methane monooxygenase (pMMO) from M. capsulatus (Bath).

Sub-3 Å Cryo-EM Structures of Necrosis Virus Particles via the Use of Multipurpose TEM with Electron Counting Camera.

During this global pandemic, cryo-EM has made a great impact on the structure determination of COVID-19 proteins. However, nearly all high-resolution results are based on data acquired on state-of-the-art microscopes where their availability is restricted to a number of centers across the globe with the studies on infectious viruses being further regulated or forbidden. One potential remedy is to employ multipurpose microscopes.

Activation of Prp28 ATPase by phosphorylated Npl3 at a critical step of spliceosome remodeling.

Splicing, a key step in the eukaryotic gene-expression pathway, converts precursor messenger RNA (pre-mRNA) into mRNA by excising introns and ligating exons. This task is accomplished by the spliceosome, a macromolecular machine that must undergo sequential conformational changes to establish its active site. Each of these major changes requires a dedicated DExD/H-box ATPase, but how these enzymes are activated remain obscure.

Pre-pro is a fast pre-processor for single-particle cryo-EM by enhancing 2D classification.

2D classification plays a pivotal role in analyzing single particle cryo-electron microscopy images. Here, we introduce a simple and loss-less pre-processor that incorporates a fast dimension-reduction (2SDR) de-noiser to enhance 2D classification. By implementing this 2SDR pre-processor prior to a representative classification algorithm like RELION and ISAC, we compare the performances with and without the pre-processor.

NMR assignments of protrusion domain of capsid protein from dragon grouper nervous necrosis virus.

Nervous necrosis virus (NNV) is a non-enveloped virus that causes massive mortality in aquaculture fish production worldwide. Recently X-ray crystallography and single particle cryo-EM have independently determined the icosahedral capsid of NNV to near-atomic resolutions to show the capsid protein is composed of a S-domain (shell) and a P-domain (protrusion) connected by a linker. However, the structure of the spike on NNV capsid made of trimeric P-domains was poorly resolved by cryo-EM.

Deriving a sub-nanomolar affinity peptide from TAP to enable smFRET analysis of RNA polymerase II complexes.

Our capability to visualize protein complexes such as RNA polymerase II (pol II) by single-molecule imaging techniques has largely been hampered by the absence of a simple bio-orthogonal approach for selective labeling with a fluorescent probe. Here, we modify the existing calmodulin-binding peptide (CBP) in the widely used Tandem Affinity Purification (TAP) tag to endow it with a high affinity for calmodulin (CaM) and use dye-CaM to conduct site-specific labeling of pol II. To demonstrate the single molecule applicability of this approach, we labeled the C-terminus of the Rpb9 subunit of pol II with donor-CaM and a site in TFIIF with an acceptor to generate a FRET (fluorescence resonance energy transfer) pair in the pol II-TFIIF complex.

Characterization of a lytic vibriophage VP06 of Vibrio parahaemolyticus.

Vibrio parahaemolyticus is a human enteropathogenic bacterium and is also pathogenic to shrimp and finfish. In a search for a biocontrol agent for V. parahaemolyticus and other pathogenic Vibrio species, a lytic phage VP06 was isolated from oyster using V.

Development of a high-growth enterovirus 71 vaccine candidate inducing cross-reactive neutralizing antibody responses.

Although Enterovirus 71 (EV71) has only one serotype based on serum neutralization tests using hyperimmune animal antisera, three major genogroups (A, B and C) including eleven genotypes (A, B1-B2, and C1-C5) can be well classified based on phylogenetic analysis. Since 1997, large-scale EV71 epidemics occurred cyclically with different genotypes in the Asia-Pacific region. Therefore, development of EV71 vaccines is a national priority in several Asian countries.

Free-electron-laser coherent diffraction images of individual drug-carrying liposome particles in solution.

Using the excellent performances of a SACLA (RIKEN/HARIMA, Japan) X-ray free electron laser (X-FEL), coherent diffraction imaging (CDI) was used to detect individual liposome particles in water, with or without inserted doxorubicin nanorods. This was possible because of the electron density differences between the carrier, the liposome, and the drug. The result is important since liposome nanocarriers at present dominate drug delivery systems.

The conserved AU dinucleotide at the 5' end of nascent U1 snRNA is optimized for the interaction with nuclear cap-binding-complex.

Splicing is initiated by a productive interaction between the pre-mRNA and the U1 snRNP, in which a short RNA duplex is established between the 5' splice site of a pre-mRNA and the 5' end of the U1 snRNA. A long-standing puzzle has been why the AU dincucleotide at the 5'-end of the U1 snRNA is highly conserved, despite the absence of an apparent role in the formation of the duplex. To explore this conundrum, we varied this AU dinucleotide into all possible permutations and analyzed the resulting molecular consequences.

Hybrid electron microscopy-FRET imaging localizes the dynamical C-terminus of Tfg2 in RNA polymerase II-TFIIF with nanometer precision.

TFIIF-a general transcription factor comprising two conserved subunits can associate with RNA polymerase II (RNAPII) tightly to regulate the synthesis of messenger RNA in eukaryotes. Herein, a hybrid method that combines electron microscopy (EM) and Förster resonance energy transfer (FRET) is described and used to localize the C-terminus of the second TFIIF subunit (Tfg2) in the architecture of RNAPII-TFIIF. In the first stage, a poly-histidine tag appended to the Tfg2 C-terminus was labeled with nickel-NTA nanogold and a seven-step single particle EM protocol was devised to obtain the region accessible by the nanogold in 3D, suggesting the Tfg2 C-terminus is proximal to the clamp of RNAPII.

Regulation of mammalian transcription by Gdown1 through a novel steric crosstalk revealed by cryo-EM.

In mammals, a distinct RNA polymerase II form, RNAPII(G) contains a novel subunit Gdown1 (encoded by POLR2M), which represses gene activation, only to be reversed by the multisubunit Mediator co-activator. Here, we employed single-particle cryo-electron microscopy (cryo-EM) to disclose the architectures of RNAPII(G), RNAPII and RNAPII in complex with the transcription initiation factor TFIIF, all to ~19 Å. Difference analysis mapped Gdown1 mostly to the RNAPII Rpb5 shelf-Rpb1 jaw, supported by antibody labelling experiments.

A Lon-like protease with no ATP-powered unfolding activity.

Lon proteases are a family of ATP-dependent proteases involved in protein quality control, with a unique proteolytic domain and an AAA(+) (ATPases associated with various cellular activities) module accommodated within a single polypeptide chain. They were classified into two types as either the ubiquitous soluble LonA or membrane-inserted archaeal LonB. In addition to the energy-dependent forms, a number of medically and ecologically important groups of bacteria encode a third type of Lon-like proteins in which the conserved proteolytic domain is fused to a large N-terminal fragment lacking canonical AAA(+) motifs.

Toward automated denoising of single molecular Förster resonance energy transfer data.

A wide-field two-channel fluorescence microscope is a powerful tool as it allows for the study of conformation dynamics of hundreds to thousands of immobilized single molecules by Förster resonance energy transfer (FRET) signals. To date, the data reduction from a movie to a final set containing meaningful single-molecule FRET (smFRET) traces involves human inspection and intervention at several critical steps, greatly hampering the efficiency at the post-imaging stage. To facilitate the data reduction from smFRET movies to smFRET traces and to address the noise-limited issues, we developed a statistical denoising system toward fully automated processing.

Roles of cysteines Cys115 and Cys201 in the assembly and thermostability of grouper betanodavirus particles.

The virus-like particle (VLP) assembled from capsid subunits of the dragon grouper nervous necrosis virus (DGNNV) is very similar to its native T = 3 virion. In order to investigate the effects of four cysteine residues in the capsid polypeptide on the assembly/dissociation pathways of DGNNV virions, we recombinantly cloned mutant VLPs by mutating each cysteine to destroy the specific disulfide linkage as compared with thiol reduction to destroy all S-S bonds. The mutant VLPs of C187A and C331A mutations were similar to wild-type VLPs (WT-VLPs); hence, the effects of Cys187 and Cys331 on the particle formation and thermostability were presumably negligible.

Zernike phase plate cryoelectron microscopy facilitates single particle analysis of unstained asymmetric protein complexes.

Single particle reconstruction from cryoelectron microscopy images, though emerging as a powerful means in structural biology, is faced with challenges as applied to asymmetric proteins smaller than megadaltons due to low contrast. Zernike phase plate can improve the contrast by restoring the microscope contrast transfer function. Here, by exploiting simulated Zernike and conventional defocused cryoelectron microscope images with noise characteristics comparable to those of experimental data, we quantified the efficiencies of the steps in single particle analysis of ice-embedded RNA polymerase II (500 kDa), transferrin receptor complex (290 kDa), and T7 RNA polymerase lysozyme (100 kDa).

Dibenzo[f,h]thieno[3,4-b] quinoxaline-based small molecules for efficient bulk-heterojunction solar cells.

Two isomeric compounds (1 and 2) containing a dibenzo[f,h]thieno[3,4-b]quinoxaline core and two peripheral arylamines were synthesized. Solution-processed bulk heterojunction (BHJ) solar cells based on these sensitizers and [6,6]-phenyl-C61-butyric acid methyl ester (PCBM) are reported. The cell fabricated from 1/67 wt % of PCBM exhibited a high power conversion efficiency of 1.

Phase TEM for biological imaging utilizing a Boersch electrostatic phase plate: theory and practice.

A Boersch electrostatic phase plate (BEPP) used in a transmission electron microscope (TEM) system can provide tuneable phase shifts and overcome the low contrast problem for biological imaging. Theoretically, a pure phase image with a high phase contrast can be obtained using a BEPP. However, a currently available TEM system utilizing a BEPP cannot achieve sufficiently high phase efficiency for biological imaging, owing to the practical conditions.

Mapping RNA exit channel on transcribing RNA polymerase II by FRET analysis.

A simple genetic tag-based labeling method that permits specific attachment of a fluorescence probe near the C terminus of virtually any subunit of a protein complex is implemented. Its immediate application to yeast RNA polymerase II (pol II) enables us to test various hypotheses of RNA exit channel by using fluorescence resonance energy transfer (FRET) analysis. The donor dye is labeled on a site near subunit Rpb3 or Rpb4, and the acceptor dye is attached to the 5' end of RNA transcript in the pol II elongation complex.

Role of the DxxDxD motif in the assembly and stability of betanodavirus particles.

Piscine betanodavirus possesses a bipartite genome of single-stranded (+)RNAs. RNA2 cDNA of dragon grouper nervous necrosis virus (DGNNV) has been expressed previously to form virus-like particles (VLPs), which are highly similar to the native virion. Experiments with calcium-chelating or reducing/oxidizing reagents showed that the DGNNV VLPs required only calcium for particle assembly.

Study of mean absorptive potential using Lenz model: toward quantification of phase contrast from an electrostatic phase plate.

An electrostatic phase plate can provide better phase contrast, a fact that plays a promising role for the high-resolution observation of specimens containing light elements. However, in order to quantify the "phase" contrast from images recorded using the phase plate, the "absorption" (or scattering) contrast arising from electrons scattered elastically and inelastically outside of the phase-plate ring must be analyzed. Angular distributions of the elastic and inelastic scattering are predicted using the Lenz model.