positive stranded viral RNA replication / host cell nucleolus / stringent response / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / positive regulation of ribosome biogenesis / endoribonuclease inhibitor activity ...positive stranded viral RNA replication / host cell nucleolus / stringent response / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / misfolded RNA binding / Group I intron splicing / RNA folding / transcriptional attenuation / positive regulation of ribosome biogenesis / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / translational termination / negative regulation of cytoplasmic translation / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / mRNA regulatory element binding translation repressor activity / assembly of large subunit precursor of preribosome / positive regulation of RNA splicing / ribosome assembly / cytosolic ribosome assembly / response to reactive oxygen species / regulation of cell growth / picornain 3C / DNA-templated transcription termination / T=pseudo3 icosahedral viral capsid / response to radiation / maintenance of translational fidelity / host cell cytoplasmic vesicle membrane / mRNA 5'-UTR binding / ribosome biogenesis / large ribosomal subunit / transferase activity / ribosome binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / channel activity / small ribosomal subunit / 5S rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / small ribosomal subunit rRNA binding / monoatomic ion transmembrane transport / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / RNA helicase activity / negative regulation of translation / rRNA binding / RNA helicase / ribosome / structural constituent of ribosome / symbiont-mediated suppression of host gene expression / translation / symbiont entry into host cell / ribonucleoprotein complex / viral translational frameshifting / symbiont-mediated activation of host autophagy / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / response to antibiotic / RNA-directed RNA polymerase activity / negative regulation of DNA-templated transcription / DNA-templated transcription / mRNA binding / virion attachment to host cell / structural molecule activity / ATP hydrolysis activity / proteolysis / DNA binding / RNA binding / zinc ion binding / ATP binding / metal ion binding / membrane / cytosol / cytoplasm Similarity search - Function
Leader peptide, picornavirus / Viral leader polypeptide zinc finger / Virion protein N terminal domain / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 ...Leader peptide, picornavirus / Viral leader polypeptide zinc finger / Virion protein N terminal domain / Capsid protein VP4, Picornavirus / Viral protein VP4 subunit / Capsid protein VP4 superfamily, Picornavirus / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Ribosomal protein L10, eubacterial, conserved site / Ribosomal protein L10 signature. / Ribosomal protein L10 / Ribosomal protein S21, conserved site / Ribosomal protein S21 signature. / Ribosomal protein L11, bacterial-type / Ribosomal protein L25, short-form / Ribosomal protein S14, bacterial/plastid / Ribosomal protein L31 type A / Ribosomal protein S21 superfamily / Ribosomal protein S16, conserved site / Ribosomal protein S16 signature. / Ribosomal protein S21 / Ribosomal protein L31 signature. / Picornavirus coat protein / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein S21 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L10-like domain superfamily / Ribosomal protein L10P / Ribosomal protein L10 / : / Ribosomal protein L16 signature 1. / Ribosomal protein L21, conserved site / Ribosomal protein L21 signature. / Ribosomal protein L16 signature 2. / Ribosomal protein L16, conserved site / Ribosomal protein L6, conserved site / Ribosomal protein L6 signature 1. / : / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / Ribosomal protein L17 signature. / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, RNA binding domain / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L36 signature. / Ribosomal protein L32p, bacterial type / Ribosomal protein L28/L24 superfamily / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / Ribosomal protein L28 / Ribosomal protein L35, non-mitochondrial / : / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / : / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Ribosomal protein S19, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein L6, bacterial-type / Ribosomal protein S13, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein L36 / Ribosomal protein S4, bacterial-type / Ribosomal protein L36 superfamily / Ribosomal protein L36 / 30S ribosomal protein S17 / Ribosomal protein S5, bacterial-type / Ribosomal protein L19, conserved site / Ribosomal protein L19 signature. Similarity search - Domain/homology
50S ribosomal protein L10 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS13 ...50S ribosomal protein L10 / Small ribosomal subunit protein bS18 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein uS19 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS17 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS5 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS9 / Small ribosomal subunit protein uS15 / Small ribosomal subunit protein bS21 / Small ribosomal subunit protein bS16 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Large ribosomal subunit protein bL36A / Large ribosomal subunit protein bL9 / Small ribosomal subunit protein uS12 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein bL17 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein uL18 / Genome polyprotein / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL5 / Large ribosomal subunit protein bL25 / Large ribosomal subunit protein bL31 Similarity search - Component
Journal: Nat Commun / Year: 2021 Title: Structural and molecular basis for Cardiovirus 2A protein as a viral gene expression switch. Authors: Chris H Hill / Lukas Pekarek / Sawsan Napthine / Anuja Kibe / Andrew E Firth / Stephen C Graham / Neva Caliskan / Ian Brierley / Abstract: Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we ...Programmed -1 ribosomal frameshifting (PRF) in cardioviruses is activated by the 2A protein, a multi-functional virulence factor that also inhibits cap-dependent translational initiation. Here we present the X-ray crystal structure of 2A and show that it selectively binds to a pseudoknot-like conformation of the PRF stimulatory RNA element in the viral genome. Using optical tweezers, we demonstrate that 2A stabilises this RNA element, likely explaining the increase in PRF efficiency in the presence of 2A. Next, we demonstrate a strong interaction between 2A and the small ribosomal subunit and present a cryo-EM structure of 2A bound to initiated 70S ribosomes. Multiple copies of 2A bind to the 16S rRNA where they may compete for binding with initiation and elongation factors. Together, these results define the structural basis for RNA recognition by 2A, show how 2A-mediated stabilisation of an RNA pseudoknot promotes PRF, and reveal how 2A accumulation may shut down translation during virus infection.
History
Deposition
Mar 17, 2021
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Header (metadata) release
Dec 15, 2021
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Map release
Dec 15, 2021
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Update
Mar 19, 2025
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Current status
Mar 19, 2025
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : Initiated 70S ribosome in complex with 2A protein from encephalom...
Entire
Name: Initiated 70S ribosome in complex with 2A protein from encephalomyocarditis virus (EMCV)
Components
Complex: Initiated 70S ribosome in complex with 2A protein from encephalomyocarditis virus (EMCV)
Protein or peptide: 50S ribosomal protein L2
Protein or peptide: 50S ribosomal protein L3
Protein or peptide: 50S ribosomal protein L4
Protein or peptide: 50S ribosomal protein L5
Protein or peptide: 50S ribosomal protein L6
Protein or peptide: 50S ribosomal protein L9
Protein or peptide: 50S ribosomal protein L10
Protein or peptide: 50S ribosomal protein L11
Protein or peptide: 50S ribosomal protein L13
Protein or peptide: 50S ribosomal protein L14
Protein or peptide: 50S ribosomal protein L15
Protein or peptide: 50S ribosomal protein L16
Protein or peptide: 50S ribosomal protein L17
Protein or peptide: 50S ribosomal protein L18
Protein or peptide: 50S ribosomal protein L19
Protein or peptide: 50S ribosomal protein L20
Protein or peptide: 50S ribosomal protein L21
Protein or peptide: 50S ribosomal protein L22
Protein or peptide: 50S ribosomal protein L23
Protein or peptide: 50S ribosomal protein L24
Protein or peptide: 50S ribosomal protein L25
Protein or peptide: 50S ribosomal protein L27
Protein or peptide: 50S ribosomal protein L28
Protein or peptide: 50S ribosomal protein L29
Protein or peptide: 50S ribosomal protein L30
RNA: 23S ribosomal RNA
RNA: 16S ribosomal RNA
RNA: 5S ribosomal RNA
Protein or peptide: 50S ribosomal protein L31
Protein or peptide: 50S ribosomal protein L32
Protein or peptide: 50S ribosomal protein L33
Protein or peptide: 50S ribosomal protein L34
Protein or peptide: 50S ribosomal protein L35
Protein or peptide: 50S ribosomal protein L36
Protein or peptide: 30S ribosomal protein S2
Protein or peptide: 30S ribosomal protein S3
Protein or peptide: 30S ribosomal protein S4
Protein or peptide: 30S ribosomal protein S5
Protein or peptide: 30S ribosomal protein S6
Protein or peptide: 30S ribosomal protein S7
Protein or peptide: 30S ribosomal protein S8
Protein or peptide: 30S ribosomal protein S9
Protein or peptide: 30S ribosomal protein S10
Protein or peptide: 30S ribosomal protein S11
Protein or peptide: 30S ribosomal protein S12
Protein or peptide: 30S ribosomal protein S13
Protein or peptide: 30S ribosomal protein S14
Protein or peptide: 30S ribosomal protein S15
Protein or peptide: 30S ribosomal protein S16
Protein or peptide: 30S ribosomal protein S17
Protein or peptide: 30S ribosomal protein S18
Protein or peptide: 30S ribosomal protein S19
Protein or peptide: 30S ribosomal protein S20
Protein or peptide: 30S ribosomal protein S21
RNA: mRNA
Protein or peptide: Protein 2A
RNA: fMet-NH-tRNA(fMet)
Ligand: MAGNESIUM ION
Ligand: N-FORMYLMETHIONINE
Ligand: ZINC ION
Ligand: water
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Supramolecule #1: Initiated 70S ribosome in complex with 2A protein from encephalom...
Supramolecule
Name: Initiated 70S ribosome in complex with 2A protein from encephalomyocarditis virus (EMCV) type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#28, #30-#57 Details: 70S ribosome subunits and initiator fMet-tRNA purified from E.coli. mRNA template was generated by in vitro transcription EMCV 2A protein was recombinantly expressed in E.coli
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Macromolecule #1: 50S ribosomal protein L2
Macromolecule
Name: 50S ribosomal protein L2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Name: mRNA / type: rna / ID: 56 Details: EMCV frameshift site flanked by the bacterial 5' UTR with Shine-Dalgarno sequence and 18 nt downstream region of the putative structure Number of copies: 1
Details: Initiated 70S ribosomes in 50 mM Tris-HCl pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2 were diluted tenfold into 20 mM HEPES pH 7.5, 100 mM potassium acetate, 1.5 mM MgCl2, 2.0 mM DTT. 2A ...Details: Initiated 70S ribosomes in 50 mM Tris-HCl pH 7.5, 70 mM NH4Cl, 30 mM KCl, 7 mM MgCl2 were diluted tenfold into 20 mM HEPES pH 7.5, 100 mM potassium acetate, 1.5 mM MgCl2, 2.0 mM DTT. 2A protein was dialysed (3K MWCO, 277K, 16 h) into the same buffer. Crosslinking reactions of 50 microliters comprising 75 nM ribosomes, 3.0 micromolar 2A and 2.0 mM bis(sulfosuccinimidyl)suberate (BS3) were performed on ice (30 min) immediately prior to grid preparation.
Grid
Model: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
Vitrification
Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV Details: Quantifoil R 2/2 400-mesh copper supports were coated with an additional ~ 60 angstrom layer of amorphous, evaporated carbon by flotation and thoroughly dried before use. Grids were made ...Details: Quantifoil R 2/2 400-mesh copper supports were coated with an additional ~ 60 angstrom layer of amorphous, evaporated carbon by flotation and thoroughly dried before use. Grids were made hydrophilic by glow-discharge in air for 30 s. Three microliters of crosslinking reaction was applied to grids which were then blotted for 4.5 s and vitrified by plunging into liquid ethane using a Vitrobot MK IV (FEI) at 277K, 100% relative humidity..
Details
75 nM ribosomes, 3.0 micromolar 2A crosslinked with 2.0 mM bis(sulfosuccinimidyl)suberate (BS3)
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 1 / Number real images: 5730 / Average exposure time: 0.59 sec. / Average electron dose: 54.4 e/Å2 Details: Images were collected as 23-frame movies. Autofocus every 10 micrometres, no drift measurement, 7 sec delay after stage shift and 3 sec delay after image shift
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Movie frames were aligned and a dose-weighted average calculated with MotionCor2
Particle selection
Number selected: 820475 Details: Reference-free autopicking of particles was performed using the Laplacian-of-Gaussian function in Relion (200 - 250 angstrom diameter)
Details: Map was low-pass filtered to 80 angstroms resolution
Final reconstruction
Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.66 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 120749
Initial angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classification
Number classes: 6 / Avg.num./class: 20124 / Software - Name: RELION (ver. 3.1) Details: Focussed classification with signal subtraction and local angular searches was performed to separate particles based on 2A occupancy at the factor binding site
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