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- PDB-7bny: Structure of 2A protein from encephalomyocarditis virus (EMCV) -

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Basic information

Entry
Database: PDB / ID: 7bny
TitleStructure of 2A protein from encephalomyocarditis virus (EMCV)
ComponentsGenome polyprotein
KeywordsVIRAL PROTEIN / EMCV / cardiovirus / 2A / picornavirus / frameshifting / PRF / RNA-binding protein / protein-mediated frameshifting / ribosome-binding protein / beta-shell
Function / homology
Function and homology information


positive stranded viral RNA replication / host cell nucleolus / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / protein complex oligomerization / monoatomic ion channel activity / symbiont-mediated suppression of host gene expression / RNA helicase activity ...positive stranded viral RNA replication / host cell nucleolus / symbiont-mediated suppression of host cytoplasmic pattern recognition receptor signaling pathway via inhibition of RIG-I activity / picornain 3C / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / protein complex oligomerization / monoatomic ion channel activity / symbiont-mediated suppression of host gene expression / RNA helicase activity / RNA helicase / symbiont entry into host cell / induction by virus of host autophagy / RNA-directed RNA polymerase / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / virus-mediated perturbation of host defense response / DNA-templated transcription / virion attachment to host cell / structural molecule activity / ATP hydrolysis activity / proteolysis / RNA binding / ATP binding / membrane / metal ion binding
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 / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral ...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 / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) / Picornavirus capsid / picornavirus capsid protein / Helicase, superfamily 3, single-stranded RNA virus / Superfamily 3 helicase of positive ssRNA viruses domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / Picornavirus/Calicivirus coat protein / Viral coat protein subunit / RNA-directed RNA polymerase, C-terminal domain / Viral RNA-dependent RNA polymerase / Reverse transcriptase/Diguanylate cyclase domain / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase S1, PA clan, chymotrypsin-like fold / Peptidase S1, PA clan / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
Biological speciesMengo encephalomyocarditis virus
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 2.62 Å
AuthorsHill, C.H. / Napthine, S. / Pekarek, L. / Kibe, A. / Firth, A.E. / Graham, S.C. / Caliskan, N. / Brierley, I.
Funding support United Kingdom, European Union, 3items
OrganizationGrant numberCountry
Wellcome Trust202797/Z/16/Z United Kingdom
Wellcome Trust098406/Z/12/B United Kingdom
European Communitys Seventh Framework ProgrammeBIOSTRUCT-X (Contract No. 283570)European Union
CitationJournal: 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
DepositionJan 22, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 8, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Genome polyprotein
B: Genome polyprotein
C: Genome polyprotein
D: Genome polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)72,18913
Polymers71,3254
Non-polymers8659
Water2,648147
1
A: Genome polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)18,1194
Polymers17,8311
Non-polymers2883
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Genome polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)18,1194
Polymers17,8311
Non-polymers2883
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Genome polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)17,9272
Polymers17,8311
Non-polymers961
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
4
D: Genome polyprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)18,0233
Polymers17,8311
Non-polymers1922
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)91.560, 91.560, 316.390
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number180
Space group name H-MP6222
Space group name HallP622(x,y,z+1/3)
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/3
#3: y,-x+y,z+2/3
#4: -y,x-y,z+2/3
#5: -x+y,-x,z+1/3
#6: x-y,-y,-z
#7: -x,-x+y,-z+1/3
#8: -x,-y,z
#9: y,x,-z+2/3
#10: -y,-x,-z+2/3
#11: -x+y,y,-z
#12: x,x-y,-z+1/3
Components on special symmetry positions
IDModelComponents
11B-1134-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 12 through 27 or resid 29...
d_2ens_1(chain "C" and (resid 12 through 27 or resid 29...
d_3ens_1(chain "D" and (resid 12 through 27 or resid 29...

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1PROALAA3 - 18
d_12ens_1ILEARGA20 - 22
d_13ens_1LEULEUA26 - 71
d_14ens_1ALALYSA73 - 85
d_15ens_1ILEVALA87 - 95
d_16ens_1LYSVALA97 - 106
d_17ens_1GLYGLYA108 - 119
d_18ens_1PHEPHEA121
d_19ens_1LEUHISA124 - 127
d_21ens_1PROALAC1 - 16
d_22ens_1ILEARGC18 - 20
d_23ens_1LEULEUC24 - 69
d_24ens_1ALALYSC71 - 83
d_25ens_1ILEVALC85 - 93
d_26ens_1LYSVALC95 - 104
d_27ens_1GLYGLYC106 - 117
d_28ens_1PHEPHEC119
d_29ens_1LEUHISC122 - 125
d_31ens_1PROALAD1 - 16
d_32ens_1ILEARGD18 - 20
d_33ens_1LEULEUD24 - 69
d_34ens_1ALALYSD72 - 84
d_35ens_1ILEVALD86 - 94
d_36ens_1LYSVALD96 - 105
d_37ens_1GLYGLYD107 - 118
d_38ens_1PHEPHED120
d_39ens_1LEUHISD123 - 126

NCS oper:
IDCodeMatrixVector
1given(-0.984987454313, -0.145097959903, 0.0935216385536), (0.0686519296025, -0.826329306615, -0.558987289293), (0.158387586028, -0.544175026128, 0.823885255075)123.388864583, -6.57599353252, -5.95920649622
2given(-0.527033884377, 0.823993242441, 0.208015434834), (0.822809379067, 0.433499858812, 0.367508636811), (0.21264997164, 0.364846555172, -0.906458482635)99.0726092296, -65.7526724424, 34.7832709371

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Components

#1: Protein
Genome polyprotein


Mass: 17831.182 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mengo encephalomyocarditis virus / Plasmid: pOPTnH / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): pLysS
References: UniProt: P12296, RNA helicase, picornain 3C, RNA-directed RNA polymerase
#2: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 147 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.07 Å3/Da / Density % sol: 59.97 %
Crystal growTemperature: 294 K / Method: vapor diffusion, sitting drop / pH: 5.7
Details: Purified EMCV 2A was concentrated to 5.9 mg/ml in 10 mM HEPES pH 7.9, 1.0 M NaCl, 2.0 mM DTT Drops were prepared by mixing 200 nL protein and 200 nL crystallization buffer: 0.625 M ammonium ...Details: Purified EMCV 2A was concentrated to 5.9 mg/ml in 10 mM HEPES pH 7.9, 1.0 M NaCl, 2.0 mM DTT Drops were prepared by mixing 200 nL protein and 200 nL crystallization buffer: 0.625 M ammonium sulfate, 0.15 M tri-sodium citrate pH 5.7

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Data collection

Diffraction
IDMean temperature (K)Ambient temp detailsCrystal-IDSerial crystal experiment
1100nitrogen cryostream1N
2100nitrogen cryostream1N
Diffraction source
SourceSiteBeamlineIDWavelength (Å)
SYNCHROTRONDiamond I0310.97958
SYNCHROTRONDiamond I0420.97635, 0.97965, 0.97974
Detector
TypeIDDetectorDate
DECTRIS PILATUS 6M1PIXELJun 24, 2018
DECTRIS PILATUS 6M-F2PIXELSep 21, 2018
Radiation
IDMonochromatorProtocolMonochromatic (M) / Laue (L)Scattering typeWavelength-ID
1double-crystalSINGLE WAVELENGTHMx-ray1
2double-crystalMADMx-ray2
Radiation wavelength
IDWavelength (Å)Relative weight
10.979581
20.976351
30.979651
40.979741
ReflectionResolution: 2.62→43.91 Å / Num. obs: 24668 / % possible obs: 100 % / Redundancy: 18.9 % / Biso Wilson estimate: 54.9 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.227 / Rpim(I) all: 0.053 / Net I/σ(I): 11.5
Reflection shellResolution: 2.62→2.67 Å / Redundancy: 19.4 % / Rmerge(I) obs: 2.766 / Mean I/σ(I) obs: 1 / Num. unique obs: 1179 / CC1/2: 0.728 / Rpim(I) all: 0.64 / % possible all: 99.8

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Processing

Software
NameVersionClassification
GDAdata collection
xia2data reduction
XDSdata reduction
Aimlessdata scaling
SHARPphasing
SHELXDphasing
PHENIX1.18.1_3865refinement
ARP/wARPmodel building
Cootmodel building
RefinementMethod to determine structure: MAD / Resolution: 2.62→43.91 Å / SU ML: 0.3519 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 28.5018
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2511 1207 4.91 %
Rwork0.2254 23361 -
obs0.2267 24568 99.66 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 75.03 Å2
Refinement stepCycle: LAST / Resolution: 2.62→43.91 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4384 0 45 147 4576
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00334582
X-RAY DIFFRACTIONf_angle_d0.74076235
X-RAY DIFFRACTIONf_chiral_restr0.0465658
X-RAY DIFFRACTIONf_plane_restr0.0068785
X-RAY DIFFRACTIONf_dihedral_angle_d25.61041656
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AX-RAY DIFFRACTIONTorsion NCS1.01832727609
ens_1d_3AX-RAY DIFFRACTIONTorsion NCS1.09386302886
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.62-2.730.37031330.34042499X-RAY DIFFRACTION99.4
2.73-2.850.3431250.32622534X-RAY DIFFRACTION99.48
2.85-30.37621270.3082515X-RAY DIFFRACTION99.29
3-3.190.30561290.27312545X-RAY DIFFRACTION99.52
3.19-3.430.21551290.23332565X-RAY DIFFRACTION99.93
3.43-3.780.25371380.21212585X-RAY DIFFRACTION99.82
3.78-4.320.22571410.18972599X-RAY DIFFRACTION99.85
4.33-5.450.20071280.17972661X-RAY DIFFRACTION99.75
5.45-43.910.25571570.2332858X-RAY DIFFRACTION99.9
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.27895927613-0.260963278595-0.2828777750433.19726998419-0.1518874807596.42903998405-0.0759608361783-0.002503873797540.0794135857304-0.112406678794-0.09675303058580.359077864278-0.164899231971-0.6768664633460.1314321124960.291164450060.0236741092083-0.07921562173370.400041791507-0.01650097096230.41512801482377.31399149455.235357539440.9533669547
22.54988432435-1.14783024579-0.1507770681614.45778457306-0.9767442037913.89867046752-0.0386453203337-0.1159998720280.06908888005420.0231551712243-0.043376238046-0.0353066286240.37225436903-0.1831025412620.06846501648120.331063994945-0.0590729460910.03074125319360.590309215230.03774543506470.41668385280454.1805551577-21.98757330729.95984293311
35.78490244369-0.730325094308-0.7819631364264.66606480863-1.06627756364.362143259760.024939486024-0.76664334299-0.3872431774280.160578704386-0.175024034478-0.1432957662510.5553116404060.01673664857260.1247232624190.651900894467-0.1970478531280.01690747601280.7312968566080.09462827118880.38341119607549.5254570554-28.405690288436.5586139947
45.09191104189-0.847217242164-0.2782957337774.66178080623-0.1281619789284.310208904710.06353465414730.29220761087-0.05630357357150.0323464898804-0.242660654304-0.430874089151-0.253224378610.6024732943070.1575114410180.297894348341-0.094326451943-0.07400799454690.6263675133670.1411269304540.46024847911771.012681298315.550743823216.7582007129
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11(chain 'A' and resid 10 through 143)AA10 - 1411 - 132
22(chain 'B' and resid 2 through 140)BB2 - 1401 - 140
33(chain 'C' and resid 12 through 137)CC12 - 1371 - 126
44(chain 'D' and resid 12 through 137)DD12 - 1371 - 127

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