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- PDB-6gv4: High-resolution Cryo-EM of Fab-labeled human parechovirus 3 -

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Basic information

Entry
Database: PDB / ID: 6gv4
TitleHigh-resolution Cryo-EM of Fab-labeled human parechovirus 3
Components
  • (AT12-015 antibody variable ...) x 2
  • RNA (5'-R(*UP*GP*GP*UP*AP*UP*UP*U)-3')
  • VP0
  • VP1
  • VP3
KeywordsVIRUS / human parechovirus / antibody / RNA
Function / homologyRNA-directed RNA polymerase, C-terminal domain / Helicase, superfamily 3, single-stranded RNA virus / Picornavirus capsid / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / RNA-directed RNA polymerase, catalytic domain / Peptidase S1, PA clan / Viral polyprotein, parechovirus P3B / Viral polyprotein, parechovirus P3A / P-loop containing nucleoside triphosphate hydrolase / Picornaviridae P3A protein ...RNA-directed RNA polymerase, C-terminal domain / Helicase, superfamily 3, single-stranded RNA virus / Picornavirus capsid / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / RNA-directed RNA polymerase, catalytic domain / Peptidase S1, PA clan / Viral polyprotein, parechovirus P3B / Viral polyprotein, parechovirus P3A / P-loop containing nucleoside triphosphate hydrolase / Picornaviridae P3A protein / Viral coat protein subunit / Picornavirus/Calicivirus coat protein / picornavirus capsid protein / 3C cysteine protease (picornain 3C) / RNA dependent RNA polymerase / RNA helicase / Helicase, superfamily 3, single-stranded DNA/RNA virus / Peptidase C3A/C3B, picornaviral / Parechovirus Genome-linked protein / RdRp of positive ssRNA viruses catalytic domain profile. / Superfamily 3 helicase of positive ssRNA viruses domain profile. / viral genome / host cell cytoplasmic vesicle membrane / pore formation by virus in membrane of host cell / integral to membrane of host cell / RNA helicase activity / viral capsid / ion channel activity / protein complex oligomerization / viral RNA genome replication / viral entry into host cell / RNA-directed 5'-3' RNA polymerase activity / cysteine-type endopeptidase activity / virion attachment to host cell / transcription, DNA-templated / structural molecule activity / RNA binding / membrane / ATP binding / Genome polyprotein
Function and homology information
Specimen sourceHomo sapiens (human)
Human parechovirus 3
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 2.8 Å resolution
AuthorsDomanska, A. / Flatt, J.W. / Jukonen, J.J.J. / Geraets, J.A. / Butcher, S.J.
CitationJournal: J. Virol. / Year: 2018
Title: 2.8 Å resolution cryo-EM structure of human parechovirus 3 in complex with Fab from a neutralizing antibody.
Authors: Aušra Domanska / Justin W Flatt / Joonas J J Jukonen / James A Geraets / Sarah J Butcher
Abstract: Human parechovirus 3 (HPeV3) infection is associated with sepsis in neonates characterized by significant immune activation and subsequent tissue damage. Strategies to limit infection have been ...Human parechovirus 3 (HPeV3) infection is associated with sepsis in neonates characterized by significant immune activation and subsequent tissue damage. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and lack of a vaccine or specific antiviral therapy. Towards the latter, we present a 2.8 Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody AT12-015 using cryo-EM and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally at high-resolution, it was possible to model a stretch of RNA inside the virion and from this identify the key features that drive and stabilize protein-RNA association during assembly. HPeV3 is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, which despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Towards this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high-resolution it was possible to precisely define the epitope that when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interaction, viral pathogenesis mechanisms, and for finding potential cures for infection and disease.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Jun 20, 2018 / Release: Nov 21, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Nov 21, 2018Structure modelrepositoryInitial release
1.1Dec 5, 2018Structure modelData collection / Database referencescitation / citation_author_citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
D: RNA (5'-R(*UP*GP*GP*UP*AP*UP*UP*U)-3')
A: VP0
B: VP1
C: VP3
H: AT12-015 antibody variable heavy
L: AT12-015 antibody variable light


Theoretical massNumber of molelcules
Total (without water)114,4156
Polyers114,4156
Non-polymers00
Water0
1
D: RNA (5'-R(*UP*GP*GP*UP*AP*UP*UP*U)-3')
A: VP0
B: VP1
C: VP3
H: AT12-015 antibody variable heavy
L: AT12-015 antibody variable light
x 60


Theoretical massNumber of molelcules
Total (without water)6,864,880360
Polyers6,864,880360
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
MethodUCSF CHIMERA 1.12_b41623.

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Components

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RNA chain , 1 types, 1 molecules D

#1: RNA chain RNA (5'-R(*UP*GP*GP*UP*AP*UP*UP*U)-3')


Mass: 2505.489 Da / Num. of mol.: 1 / Details: RNA / Source: (natural) Human parechovirus 3 / Cell line: HT29 / Organ: colon adenocarcinoma

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Protein/peptide , 3 types, 3 molecules ABC

#2: Protein/peptide VP0


Mass: 31770.135 Da / Num. of mol.: 1 / Source: (natural) Human parechovirus 3 / Cell line: HT29 / Organ: colon adenocarcinoma / References: UniProt: Q8BES5
#3: Protein/peptide VP1


Mass: 25913.127 Da / Num. of mol.: 1 / Source: (natural) Human parechovirus 3 / Cell line: HT29 / Organ: colon adenocarcinoma / References: UniProt: Q8BES5
#4: Protein/peptide VP3


Mass: 28757.551 Da / Num. of mol.: 1 / Details: polypeptide chain / Source: (natural) Human parechovirus 3 / Cell line: HT29 / Organ: colon adenocarcinoma / References: UniProt: Q8BES5

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AT12-015 antibody variable ... , 2 types, 2 molecules HL

#5: Protein/peptide AT12-015 antibody variable heavy


Mass: 13128.613 Da / Num. of mol.: 1 / Details: polypeptide chain / Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): 293T / Production host: Homo sapiens (human)
#6: Protein/peptide AT12-015 antibody variable light


Mass: 12339.752 Da / Num. of mol.: 1 / Details: polypeptide chain / Source: (gene. exp.) Homo sapiens (human) / Cell line (production host): 293T / Production host: Homo sapiens (human)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1Human parechovirus type 3 in complex with fabs from AT12-015COMPLEX1, 2, 3, 4, 5, 60MULTIPLE SOURCES
2Human parechovirus type 3VIRUS1,2,3,41NATURAL
3fabs from AT12-015COMPLEX5,61RECOMBINANT
Molecular weightValue: 7.7 MDa / Experimental value: NO
Source (natural)
IDEntity assembly IDNcbi tax IDOrganismStrain
12195055Human parechovirus 3A308/99
239606Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Details of virusEmpty: NO / Enveloped: NO / Virus isolate: STRAIN / Virus type: VIRION
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer ID
110 mMtris hydrochlorideTris-HCl1
2150 mMsodium chlorideNaCl1
31 mMmagnesium chlorideMgCL1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: ultrathin carbon-coated lacey 400-mesh copper grids (Ted Pella product #01824)
Grid material: COPPER / Grid mesh size: 400
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Chamber temperature: 295 kelvins
Details: We could not control humidity during plunging. It was ambient humidity. Blot for 1 s before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 48 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Number of grids imaged: 2 / Number of real images: 6541
EM imaging opticsPhase plate: No phase plate used / Sph aberration corrector: no Cs corrector
Image scansMovie frames/image: 18 / Used frames/image: 2-17

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Processing

EM software
IDNameVersionCategoryDetails
1RELION2.0particle selection
2RELION2.0image acquisitionRELION
7UCSF Chimera1.12model fitting
9RELION2.0initial Euler assignment
10RELION2.0final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
13MDFFmodel refinement
14Coot0.8.8model refinement
CTF correctionDetails: GCTF was used to estimate ctf / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionDetails: automatic particle selection in RELION using template generated from manually selected particles
Number of particles selected: 217212
SymmetryPoint symmetry: I
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 74927 / Number of class averages: 3 / Symmetry type: POINT
Atomic model buildingDetails: Initial model was generated in I-TASSER and SWISSMODEL using 4z92 and 4udf as reference. Initial rigid fit of the model to the map was done in UCSF Chimera. Model refinement was done in Coot and MDFF.
Ref protocol: OTHER / Ref space: REAL

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