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- EMDB-21133: Cryo-EM SPA : Structure of the PCBP2/Stem Loop IVm complex underl... -

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Basic information

Entry
Database: EMDB / ID: EMD-21133
TitleCryo-EM SPA : Structure of the PCBP2/Stem Loop IVm complex underlying the functionality of the poliovirus type I IRES.
Map dataStructure of the PCBP2/Stem Loop
Sample
  • Complex: truncated stem loop IV of the poliovirus IRES, residues, 278-398 in complex with PolyC binding protein 2
    • Protein or peptide: PolyC Binding Protein 2
    • RNA: Poliovirus (Mahoney) : truncated Stemloop IV from IRES
Biological speciesPoliovirus type 1 (strain Mahoney) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.1 Å
AuthorsWilce MCJ / Wilce JA
Funding support Australia, 1 items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)APP1161916 Australia
CitationJournal: Nucleic Acids Res / Year: 2020
Title: Structure of the PCBP2/stem-loop IV complex underlying translation initiation mediated by the poliovirus type I IRES.
Authors: Simone A Beckham / Mehdi Y Matak / Matthew J Belousoff / Hariprasad Venugopal / Neelam Shah / Naveen Vankadari / Hans Elmlund / Joseph H C Nguyen / Bert L Semler / Matthew C J Wilce / Jacqueline A Wilce /
Abstract: The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral ...The poliovirus type I IRES is able to recruit ribosomal machinery only in the presence of host factor PCBP2 that binds to stem-loop IV of the IRES. When PCBP2 is cleaved in its linker region by viral proteinase 3CD, translation initiation ceases allowing the next stage of replication to commence. Here, we investigate the interaction of PCBP2 with the apical region of stem-loop IV (SLIVm) of poliovirus RNA in its full-length and truncated form. CryoEM structure reconstruction of the full-length PCBP2 in complex with SLIVm solved to 6.1 Å resolution reveals a compact globular complex of PCBP2 interacting with the cruciform RNA via KH domains and featuring a prominent GNRA tetraloop. SEC-SAXS, SHAPE and hydroxyl-radical cleavage establish that PCBP2 stabilizes the SLIVm structure, but upon cleavage in the linker domain the complex becomes more flexible and base accessible. Limited proteolysis and REMSA demonstrate the accessibility of the linker region in the PCBP2/SLIVm complex and consequent loss of affinity of PCBP2 for the SLIVm upon cleavage. Together this study sheds light on the structural features of the PCBP2/SLIV complex vital for ribosomal docking, and the way in which this key functional interaction is regulated following translation of the poliovirus genome.
History
DepositionDec 16, 2019-
Header (metadata) releaseJan 15, 2020-
Map releaseAug 12, 2020-
UpdateAug 26, 2020-
Current statusAug 26, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21133.png

Downloads & links

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Map

FileDownload / File: emd_21133.map.gz / Format: CCP4 / Size: 11.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the PCBP2/Stem Loop
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.06869157 - 0.1436145
Average (Standard dev.)0.0004524068 (±0.0059334515)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 152.63998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z152.640152.640152.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.0690.1440.000

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Supplemental data

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Sample components

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Entire : truncated stem loop IV of the poliovirus IRES, residues, 278-398 ...

EntireName: truncated stem loop IV of the poliovirus IRES, residues, 278-398 in complex with PolyC binding protein 2
Components
  • Complex: truncated stem loop IV of the poliovirus IRES, residues, 278-398 in complex with PolyC binding protein 2
    • Protein or peptide: PolyC Binding Protein 2
    • RNA: Poliovirus (Mahoney) : truncated Stemloop IV from IRES

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Supramolecule #1: truncated stem loop IV of the poliovirus IRES, residues, 278-398 ...

SupramoleculeName: truncated stem loop IV of the poliovirus IRES, residues, 278-398 in complex with PolyC binding protein 2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Poliovirus type 1 (strain Mahoney) / Strain: Mahoney

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Macromolecule #1: PolyC Binding Protein 2

MacromoleculeName: PolyC Binding Protein 2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDTGVIEGGL NVTLTIRLLM HGKEVGSIIG KKGESVKKMR EESGARINIS EGNCPERIIT LAGPTNAIF KAFAMIIDKL EEDISSSMTN STAASRPPVT LRLVVPASQC GSLIGKGGCK I KEIRESTG AQVQVAGDML PNSTERAITI AGIPQSIIEC VKQICVVMLE ...String:
MDTGVIEGGL NVTLTIRLLM HGKEVGSIIG KKGESVKKMR EESGARINIS EGNCPERIIT LAGPTNAIF KAFAMIIDKL EEDISSSMTN STAASRPPVT LRLVVPASQC GSLIGKGGCK I KEIRESTG AQVQVAGDML PNSTERAITI AGIPQSIIEC VKQICVVMLE TLSQSPPKGV TI PYRPKPS SSPVIFAGGQ DRYSTGSDSA SFPHTTPSMC LNPDLEGPPL EAYTIQGQYA IPQ PDLTKL HQLAMQQSHF PMTHGNTGFS GIESSSPEVK GYWGLDASAQ TTSHELTIPN DLIG CIIGR QGAKINEIRQ MSGAQIKIAN PVEGSTDRQV TITGSAASIS LAQYLINVRL SSETG GMGS SLEHHHHHH

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Macromolecule #2: Poliovirus (Mahoney) : truncated Stemloop IV from IRES

MacromoleculeName: Poliovirus (Mahoney) : truncated Stemloop IV from IRES
type: rna / ID: 2
Details: Produced using in vitro transcription : T7 RiboMAX (Promega)
Source (natural)Organism: Poliovirus type 1 (strain Mahoney) / Strain: Mahoney
SequenceString:
CGCGCGUUGC GCUCAGCACU CAACCCCAGA GUGUAGCUUA GGCUGAUGAG UCUGGACAUC CCUCACCGGU GACGGUGGUC CAGGCUGCGU UGGCGGCCUA CCUAUGGCUA ACGCCAUGGG ACGCGCG

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

Concentration0.14 mg/mL
BufferpH: 7.5
Details: 5 mM HEPES-KOH, 25 mM KCl, 2 mM MgCl2, 2 mM DTT, 4 % glycerol, 0.1 mM EDTA, pH 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 2.5 second blot.
DetailsA mono disperse sample was applied

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND / Software - details: CTFFIND
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 888988
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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