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- EMDB-12635: Initiated 70S ribosome in complex with 2A protein from encephalom... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-12635 | ||||||||||||
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Title | Initiated 70S ribosome in complex with 2A protein from encephalomyocarditis virus (EMCV) | ||||||||||||
![]() | Post-processed, masked map weighted by local resolution | ||||||||||||
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![]() | 70S ribosome / initiation complex / EMCV / cardiovirus / 2A / picornavirus / frameshifting / PRF / RNA-binding protein / protein-mediated frameshifting / ribosome-binding protein / beta-shell / viral protein / RIBOSOME | ||||||||||||
Function / homology | ![]() 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 / endoribonuclease inhibitor activity / RNA-binding transcription regulator 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 / endoribonuclease inhibitor activity / RNA-binding transcription regulator activity / positive regulation of ribosome biogenesis / negative regulation of cytoplasmic translation / translational termination / DnaA-L2 complex / translation repressor activity / negative regulation of DNA-templated DNA replication initiation / ribosome assembly / mRNA regulatory element binding translation repressor activity / picornain 3C / positive regulation of RNA splicing / assembly of large subunit precursor of preribosome / T=pseudo3 icosahedral viral capsid / cytosolic ribosome assembly / response to reactive oxygen species / regulation of cell growth / DNA-templated transcription termination / host cell cytoplasmic vesicle membrane / maintenance of translational fidelity / response to radiation / ribosomal large subunit assembly / mRNA 5'-UTR binding / large ribosomal subunit / ribosome biogenesis / ribosome binding / protein complex oligomerization / monoatomic ion channel activity / ribosomal small subunit biogenesis / ribosomal small subunit assembly / symbiont-mediated suppression of host gene expression / small ribosomal subunit / small ribosomal subunit rRNA binding / transferase activity / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / tRNA binding / cytoplasmic translation / RNA helicase activity / rRNA binding / negative regulation of translation / ribosome / RNA helicase / structural constituent of ribosome / ribonucleoprotein complex / symbiont entry into host cell / induction by virus of host autophagy / translation / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / response to antibiotic / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / 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 / membrane / metal ion binding / cytoplasm / cytosol Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.66 Å | ||||||||||||
![]() | Hill CH / Napthine S | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 197.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 82.1 KB 82.1 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 15.8 KB | Display | ![]() |
Images | ![]() | 86.7 KB | ||
Masks | ![]() | 347.6 MB | ![]() | |
Filedesc metadata | ![]() | 16.8 KB | ||
Others | ![]() ![]() ![]() | 278 MB 278 MB 277.8 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 513.7 KB | Display | ![]() |
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Full document | ![]() | 513.3 KB | Display | |
Data in XML | ![]() | 15.1 KB | Display | |
Data in CIF | ![]() | 20.6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7nwtMC ![]() 7bnyC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Post-processed, masked map weighted by local resolution | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.07 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
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Projections & Slices |
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Density Histograms |
-Additional map: Unmasked map from Refine3D
File | emd_12635_additional_1.map | ||||||||||||
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Annotation | Unmasked map from Refine3D | ||||||||||||
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Density Histograms |
-Additional map: Unmasked, unfiltered half-map (1) from Refine3D
File | emd_12635_additional_2.map | ||||||||||||
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Annotation | Unmasked, unfiltered half-map (1) from Refine3D | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Unmasked, unfiltered half-map (2) from Refine3D
File | emd_12635_additional_3.map | ||||||||||||
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Annotation | Unmasked, unfiltered half-map (2) from Refine3D | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
+Entire : Initiated 70S ribosome in complex with 2A protein from encephalom...
+Supramolecule #1: Initiated 70S ribosome in complex with 2A protein from encephalom...
+Macromolecule #1: 50S ribosomal protein L2
+Macromolecule #2: 50S ribosomal protein L3
+Macromolecule #3: 50S ribosomal protein L4
+Macromolecule #4: 50S ribosomal protein L5
+Macromolecule #5: 50S ribosomal protein L6
+Macromolecule #6: 50S ribosomal protein L9
+Macromolecule #7: 50S ribosomal protein L10
+Macromolecule #8: 50S ribosomal protein L11
+Macromolecule #9: 50S ribosomal protein L13
+Macromolecule #10: 50S ribosomal protein L14
+Macromolecule #11: 50S ribosomal protein L15
+Macromolecule #12: 50S ribosomal protein L16
+Macromolecule #13: 50S ribosomal protein L17
+Macromolecule #14: 50S ribosomal protein L18
+Macromolecule #15: 50S ribosomal protein L19
+Macromolecule #16: 50S ribosomal protein L20
+Macromolecule #17: 50S ribosomal protein L21
+Macromolecule #18: 50S ribosomal protein L22
+Macromolecule #19: 50S ribosomal protein L23
+Macromolecule #20: 50S ribosomal protein L24
+Macromolecule #21: 50S ribosomal protein L25
+Macromolecule #22: 50S ribosomal protein L27
+Macromolecule #23: 50S ribosomal protein L28
+Macromolecule #24: 50S ribosomal protein L29
+Macromolecule #25: 50S ribosomal protein L30
+Macromolecule #30: 50S ribosomal protein L31
+Macromolecule #31: 50S ribosomal protein L32
+Macromolecule #32: 50S ribosomal protein L33
+Macromolecule #33: 50S ribosomal protein L34
+Macromolecule #34: 50S ribosomal protein L35
+Macromolecule #35: 50S ribosomal protein L36
+Macromolecule #36: 30S ribosomal protein S2
+Macromolecule #37: 30S ribosomal protein S3
+Macromolecule #38: 30S ribosomal protein S4
+Macromolecule #39: 30S ribosomal protein S5
+Macromolecule #40: 30S ribosomal protein S6
+Macromolecule #41: 30S ribosomal protein S7
+Macromolecule #42: 30S ribosomal protein S8
+Macromolecule #43: 30S ribosomal protein S9
+Macromolecule #44: 30S ribosomal protein S10
+Macromolecule #45: 30S ribosomal protein S11
+Macromolecule #46: 30S ribosomal protein S12
+Macromolecule #47: 30S ribosomal protein S13
+Macromolecule #48: 30S ribosomal protein S14
+Macromolecule #49: 30S ribosomal protein S15
+Macromolecule #50: 30S ribosomal protein S16
+Macromolecule #51: 30S ribosomal protein S17
+Macromolecule #52: 30S ribosomal protein S18
+Macromolecule #53: 30S ribosomal protein S19
+Macromolecule #54: 30S ribosomal protein S20
+Macromolecule #55: 30S ribosomal protein S21
+Macromolecule #57: Protein 2A
+Macromolecule #26: 23S ribosomal RNA
+Macromolecule #27: 16S ribosomal RNA
+Macromolecule #28: 5S ribosomal RNA
+Macromolecule #29: fMet-NH-tRNA(fMet)
+Macromolecule #56: mRNA
+Macromolecule #58: MAGNESIUM ION
+Macromolecule #59: N-FORMYLMETHIONINE
+Macromolecule #60: ZINC ION
+Macromolecule #61: water
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 Component:
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. | ||||||||||||||||
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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 |
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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: ![]() |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 75000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |