Different tertiary interactions create the same important 3-D features in a divergent flavivirus xrRNA

ABSTRACTDuring infection by a flavivirus (FV), cells accumulate noncoding subgenomic flavivirus RNAs (sfRNAs) that interfere with several antiviral pathways. These sfRNAs are formed by structured RNA elements in the 3′ untranslated region (UTR) of the viral genomic RNA, which block the progression of host cell exoribonucleases that have targeted the viral RNA for destruction. Previous work on these exoribonuclease-resistant RNAs (xrRNAs) from mosquito-borne FVs revealed a specific 3-dimensional fold with a unique topology in which a ring-like structure protectively encircles the 5′ end of the xrRNA. Conserved nucleotides make specific tertiary interactions that support this fold. Examination of more divergent FVs reveals differences in their 3′ UTR sequences, raising the question of whether they contain xrRNAs and if so, how they fold. To answer this, we demonstrated the presence of an authentic xrRNA in the 3′ UTR of the Tamana Bat Virus (TABV) and solved its structure by x-ray crystallography. The structure reveals conserved features from previously characterized xrRNAs, but in the TABV version these features are created through a novel set of tertiary interactions not previously seen in xrRNAs. This includes two important A-C interactions, four distinct backbone kinks, several ordered Mg2+ ions, and a C+-G-C base triple. The discovery that the same overall architecture can be achieved by very different sequences and interactions in distantly related flaviviruses provides insight into the diversity of this type of RNA and will inform searches for undiscovered xrRNAs in viruses and beyond.

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Rational synthesis of an atomically precise carboncone under mild conditions

Science Advances ◽

10.1126/sciadv.aaw0982 ◽

2019 ◽

Vol 5 (8) ◽

pp. eaaw0982 ◽

Cited By ~ 7

Author(s):

Zheng-Zhong Zhu ◽

Zuo-Chang Chen ◽

Yang-Rong Yao ◽

Cun-Hao Cui ◽

Shu-Hui Li ◽

...

Keyword(s):

Apex Angle ◽

Facile Synthesis ◽

Reaction Conditions ◽

X Ray ◽

Carbon Allotropes ◽

X Ray Crystallography ◽

Mild Conditions ◽

Research Opportunity ◽

Insight Into ◽

Structural Strain

Carboncones, a special family of all-carbon allotropes, are predicted to have unique properties that distinguish them from fullerenes, carbon nanotubes, and graphenes. Owing to the absence of methods to synthesize atomically well-defined carboncones, however, experimental insight into the nature of pure carboncones has been inaccessible. Herein, we describe a facile synthesis of an atomically well-defined carboncone[1,2] (C70H20) and its soluble penta-mesityl derivative. Identified by x-ray crystallography, the carbon skeleton is a carboncone with the largest possible apex angle. Much of the structural strain is overcome in the final step of converting the bowl-shaped precursor into the rigid carboncone under mild reaction conditions. This work provides a research opportunity for investigations of atomically precise single-layered carboncones having even higher cone walls and/or smaller apex angles.

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How big is big? Separation by conventional methods, X-ray and electronic structures of positional isomers of bis-tert-butylisocyano adduct of 2(3),9(10),16(17),23(24)-tetrachloro-3(2),10(9),17(16),24(23)-tetra(2,6-di-iso-propylphenoxy)-phthalocyaninato iron(II) complex

Journal of Porphyrins and Phthalocyanines ◽

10.1142/s1088424616500164 ◽

2016 ◽

Vol 20 (01n04) ◽

pp. 337-351 ◽

Cited By ~ 4

Author(s):

Derrick R. Anderson ◽

Pavlo V. Solntsev ◽

Hannah M. Rhoda ◽

Victor N. Nemykin

Keyword(s):

Electronic Structures ◽

Density Functional ◽

Positional Isomers ◽

Excitation Energies ◽

Functional Theory ◽

X Ray ◽

Vertical Excitation ◽

X Ray Crystallography ◽

Tddft Calculations ◽

Insight Into

A presence of bulky 2,6-di-iso-propylphenoxy groups in bis-tert-butylisocyano adduct of 2(3),9(10),16(17),23(24)-tetrachloro-3(2),10(9),17(16),24(23)-tetra(2,6-di-iso-propylphenoxy)-phthalocyaninato iron(II) complex allows separation of two individual positional isomers and a mixture of the remaining two isomers using conventional chromatography. X-ray structures of “[Formula: see text]” and “[Formula: see text]” isomers were confimed by X-ray crystallography. Density functional theory (DFT) and time-dependent DFT (TDDFT) calculations of each individual positional isomer allowed insight into their electronic structures and vertical excitation energies, which were correlated with the experimental UV-vis and MCD spectra.

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PHF13 is a molecular reader and transcriptional co-regulator of H3K4me2/3

eLife ◽

10.7554/elife.10607 ◽

2016 ◽

Vol 5 ◽

Cited By ~ 14

Author(s):

Ho-Ryun Chung ◽

Chao Xu ◽

Alisa Fuchs ◽

Andreas Mund ◽

Martin Lange ◽

...

Keyword(s):

Dna Damage Response ◽

Size Exclusion ◽

X Ray ◽

Chip Sequencing ◽

X Ray Crystallography ◽

Genome Wide ◽

Damage Response ◽

Exclusion Chromatography ◽

Insight Into ◽

Binding Cell

PHF13 is a chromatin affiliated protein with a functional role in differentiation, cell division, DNA damage response and higher chromatin order. To gain insight into PHF13's ability to modulate these processes, we elucidate the mechanisms targeting PHF13 to chromatin, its genome wide localization and its molecular chromatin context. Size exclusion chromatography, mass spectrometry, X-ray crystallography and ChIP sequencing demonstrate that PHF13 binds chromatin in a multivalent fashion via direct interactions with H3K4me2/3 and DNA, and indirectly via interactions with PRC2 and RNA PolII. Furthermore, PHF13 depletion disrupted the interactions between PRC2, RNA PolII S5P, H3K4me3 and H3K27me3 and resulted in the up and down regulation of genes functionally enriched in transcriptional regulation, DNA binding, cell cycle, differentiation and chromatin organization. Together our findings argue that PHF13 is an H3K4me2/3 molecular reader and transcriptional co-regulator, affording it the ability to impact different chromatin processes.

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Insight into the binding affinity of thiourea in the calcium binding pocket of proteinase K, through high resolution X-ray crystallography

Bioorganic Chemistry ◽

10.1016/j.bioorg.2019.103443 ◽

2020 ◽

Vol 94 ◽

pp. 103443

Author(s):

Malik Shoaib Ahmad ◽

Zeeshan Akbar ◽

M. Iqbal Choudhary

Keyword(s):

High Resolution ◽

Binding Affinity ◽

Calcium Binding ◽

Binding Pocket ◽

Proteinase K ◽

X Ray ◽

X Ray Crystallography ◽

Insight Into

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Structural insight into the Staphylococcus aureus ATP-driven exporter of virulent peptide toxins

Science Advances ◽

10.1126/sciadv.abb8219 ◽

2020 ◽

Vol 6 (40) ◽

pp. eabb8219

Author(s):

N. Zeytuni ◽

S. W. Dickey ◽

J. Hu ◽

H. T. Chou ◽

L. J. Worrall ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Bound State ◽

Broad Spectrum Antibiotic ◽

X Ray ◽

X Ray Crystallography ◽

Open Conformation ◽

Mechanistic Insight ◽

Cytolytic Peptides ◽

Peptide Toxins ◽

Insight Into

Staphylococcus aureus is a major human pathogen that has acquired alarming broad-spectrum antibiotic resistance. One group of secreted toxins with key roles during infection is the phenol-soluble modulins (PSMs). PSMs are amphipathic, membrane-destructive cytolytic peptides that are exported to the host-cell environment by a designated adenosine 5′-triphosphate (ATP)–binding cassette (ABC) transporter, the PSM transporter (PmtABCD). Here, we demonstrate that the minimal Pmt unit necessary for PSM export is PmtCD and provide its first atomic characterization by single-particle cryo-EM and x-ray crystallography. We have captured the transporter in the ATP-bound state at near atomic resolution, revealing a type II ABC exporter fold, with an additional cytosolic domain. Comparison to a lower-resolution nucleotide-free map displaying an “open” conformation and putative hydrophobic inner chamber of a size able to accommodate the binding of two PSM peptides provides mechanistic insight and sets the foundation for therapeutic design.

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Role of Computational Methods in Going beyond X-ray Crystallography to Explore Protein Structure and Dynamics

International Journal of Molecular Sciences ◽

10.3390/ijms19113401 ◽

2018 ◽

Vol 19 (11) ◽

pp. 3401 ◽

Cited By ~ 16

Author(s):

Ashutosh Srivastava ◽

Tetsuro Nagai ◽

Arpita Srivastava ◽

Osamu Miyashita ◽

Florence Tama

Keyword(s):

Protein Dynamics ◽

Computational Methods ◽

Protein Structures ◽

Three Dimensional ◽

Dimensional Structure ◽

X Ray ◽

X Ray Crystallography ◽

Insight Into

Protein structural biology came a long way since the determination of the first three-dimensional structure of myoglobin about six decades ago. Across this period, X-ray crystallography was the most important experimental method for gaining atomic-resolution insight into protein structures. However, as the role of dynamics gained importance in the function of proteins, the limitations of X-ray crystallography in not being able to capture dynamics came to the forefront. Computational methods proved to be immensely successful in understanding protein dynamics in solution, and they continue to improve in terms of both the scale and the types of systems that can be studied. In this review, we briefly discuss the limitations of X-ray crystallography in studying protein dynamics, and then provide an overview of different computational methods that are instrumental in understanding the dynamics of proteins and biomacromolecular complexes.

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Transcriptional and translational events during reovirus infection

Biochemistry and Cell Biology ◽

10.1139/o88-092 ◽

1988 ◽

Vol 66 (8) ◽

pp. 803-812 ◽

Cited By ~ 4

Author(s):

Guy Lemay

Keyword(s):

Protein Synthesis ◽

Host Cell ◽

Mrna Translation ◽

Cell Membrane Permeability ◽

Cell Protein ◽

Viral Mrna ◽

Genomic Rna ◽

Host Cell Protein ◽

Outer Capsid ◽

Viral Genomic Rna

This short review focuses on the mechanisms involved in transcription and translation in mouse L cells infected with reoviruses. The viral genomic RNA (double-stranded), retained in the inner capsid following removal of the outer capsid of the infecting virion, is transcribed by a viral polymerase. The synthesized viral mRNA is blocked at the 5′ end by a cap structure similar to the cap structure of cellular mRNA but synthesized by the viral enzymes of the inner capsid. This viral mRNA is also used as the first strand and template for the synthesis of the second strand of viral genomic RNA; the newly replicated genome is retained in an inner capsid structure to generate the progeny subviral particles. These particles are active at the transcriptional level but do not synthesize the cap, owing to the absence of the guanylyltransferase activity involved in the formation of this structure. The uncapped mRNA, or late viral mRNA, constitutes the bulk part of viral mRNA. The transcription of the viral genome is finally arrested upon addition of outer capsid proteins to obtain a mature virion. During viral multiplication, there is a gradual inhibition of host-cell protein synthesis, concomitant with stimulation of late viral mRNA translation. The two phenomena are apparently distinct. Furthermore, the inhibition of host-cell protein synthesis has been shown to be dispensable for normal virus multiplication; however, it might accelerate it. The mechanisms responsible for inhibition are still unclear but might involve modifications in the activity of cellular cap-binding proteins. This last point suggests an analogy with poliovirus infection; the two systems are thus briefly compared. Possible significance of the absence of a poly(A) tract at the 3′ end of reovirus mRNA, in contrast to the occurrence of such a sequence at the end of cellular mRNA, is also examined. Different models involving cap discrimination, competition between mRNAs, or alteration of cell membrane permeability have been proposed to explain the events observed at the translational level in reovirus-infected cells. These different models are compared. Finally, recent data implicating the viral sigma 3 capsid protein in efficient translation of late viral mRNA are discussed.

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Assembly of Severe Acute Respiratory Syndrome Coronavirus RNA Packaging Signal into Virus-Like Particles Is Nucleocapsid Dependent

Journal of Virology ◽

10.1128/jvi.79.22.13848-13855.2005 ◽

2005 ◽

Vol 79 (22) ◽

pp. 13848-13855 ◽

Cited By ~ 96

Author(s):

Ping-Kun Hsieh ◽

Shin C. Chang ◽

Chu-Chun Huang ◽

Ting-Ting Lee ◽

Ching-Wen Hsiao ◽

...

Keyword(s):

Severe Acute Respiratory Syndrome ◽

Culture Medium ◽

Binding Activity ◽

Viral Rna ◽

Structural Proteins ◽

Genomic Rna ◽

Packaging Signal ◽

Virus Like Particles ◽

N Protein ◽

Viral Genomic Rna

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm3. Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3′ noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.

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