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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 / homology
Function and homology information


T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / pore formation by virus in membrane of host cell / integral to membrane of host cell / viral capsid / protein complex oligomerization / ion channel activity / viral RNA genome replication / RNA helicase activity / cysteine-type endopeptidase activity ...T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / pore formation by virus in membrane of host cell / integral to membrane of host cell / viral capsid / protein complex oligomerization / ion channel activity / viral RNA genome replication / RNA helicase activity / cysteine-type endopeptidase activity / viral entry into host cell / RNA-directed 5'-3' RNA polymerase activity / transcription, DNA-templated / virion attachment to host cell / structural molecule activity / RNA binding / membrane / ATP binding
Helicase, superfamily 3, single-stranded DNA/RNA virus / Viral coat protein subunit / Helicase, superfamily 3, single-stranded RNA virus / Viral polyprotein, parechovirus P3A / Viral polyprotein, parechovirus P3B / Peptidase S1, PA clan / RNA-directed RNA polymerase, catalytic domain / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Picornavirus capsid / RNA-directed RNA polymerase, C-terminal domain ...Helicase, superfamily 3, single-stranded DNA/RNA virus / Viral coat protein subunit / Helicase, superfamily 3, single-stranded RNA virus / Viral polyprotein, parechovirus P3A / Viral polyprotein, parechovirus P3B / Peptidase S1, PA clan / RNA-directed RNA polymerase, catalytic domain / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Picornavirus capsid / RNA-directed RNA polymerase, C-terminal domain / Peptidase C3A/C3B, picornaviral / P-loop containing nucleoside triphosphate hydrolase / Picornavirus/Calicivirus coat protein / picornavirus capsid protein / Jelly Rolls - #20 / Jelly Rolls / Sandwich / Mainly Beta
Polyprotein / Genome polyprotein
Biological speciesHomo sapiens (human)
Human parechovirus 3
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsDomanska, A. / Flatt, J.W. / Jukonen, J.J.J. / Geraets, J.A. / Butcher, S.J.
Funding support Finland, 4items
OrganizationGrant numberCountry
Academy of Finland275199 Finland
European UnionPIEF-GA-2013-628150
European UnionPIAPP-GA-2013-612308
European Union653706
CitationJournal: J. Virol. / Year: 2019
Title: A 2.8-Angstrom-Resolution Cryo-Electron Microscopy 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 characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been ...Human parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward 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-electron microscopy (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. Human parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for 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. Toward 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 interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJun 20, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 21, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 5, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.journal_id_ASTM ..._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
Revision 1.2Feb 20, 2019Group: Data collection / Database references / Category: citation / em_admin / pdbx_database_proc / Item: _citation.journal_abbrev / _em_admin.last_update
Revision 1.3Apr 3, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.title ..._citation.journal_volume / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 2.0Jun 26, 2019Group: Atomic model / Data collection / Database references
Category: atom_site / citation_author ...atom_site / citation_author / em_admin / pdbx_database_proc
Item: _atom_site.occupancy / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

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  • Biological unit as software_defined_assembly
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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-0069
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
Polymers114,4156
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
Polymers6,864,880360
Non-polymers00
Water0
TypeNameSymmetry operationNumber
point symmetry operation60
MethodUCSF CHIMERA

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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 method: isolated from a natural source / Source: (natural) Human parechovirus 3 / Cell line: HT29 / Organ: colon adenocarcinoma

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Protein , 3 types, 3 molecules ABC

#2: Protein VP0


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


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


Mass: 28757.551 Da / Num. of mol.: 1 / Details: polypeptide chain / Source method: isolated from a natural source / Source: (natural) Human parechovirus 3 / Cell line: HT29 / Organ: colon adenocarcinoma / References: UniProt: Q8BES5, UniProt: A0A291FGQ4*PLUS

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

#5: Protein AT12-015 antibody variable heavy


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


Mass: 12339.752 Da / Num. of mol.: 1 / Details: polypeptide chain
Source method: isolated from a genetically manipulated source
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 / 3D 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-IDOrganismNcbi tax-IDStrain
12Human parechovirus 3195055A308/99
23Homo sapiens (human)9606
Source (recombinant)Organism: Homo sapiens (human)
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRION
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMtris hydrochlorideTris-HClTris1
2150 mMsodium chlorideNaClSodium chloride1
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 divisions/in.
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Chamber temperature: 295 K
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
MicroscopyModel: 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 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / 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) / Num. of grids imaged: 2 / Num. of real images: 6541
EM imaging opticsPhase plate: No phase plate used / Spherical aberration corrector: no Cs corrector
Image scansMovie frames/image: 18 / Used frames/image: 2-17

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Processing

EM software
IDNameVersionCategoryDetails
1RELION2particle selection
2RELION2image acquisitionRELION
7UCSF Chimera1.12model fitting
9RELION2initial Euler assignment
10RELION2final 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 selectionNum. of particles selected: 217212
Details: automatic particle selection in RELION using template generated from manually selected particles
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74927 / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: 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.

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