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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


RNA-protein covalent cross-linking / : / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / cytoplasmic vesicle membrane / : / viral capsid / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity ...RNA-protein covalent cross-linking / : / T=pseudo3 icosahedral viral capsid / host cell cytoplasmic vesicle membrane / cytoplasmic vesicle membrane / : / viral capsid / protein complex oligomerization / monoatomic ion channel activity / RNA helicase activity / symbiont entry into host cell / viral RNA genome replication / cysteine-type endopeptidase activity / RNA-dependent RNA polymerase activity / DNA-templated transcription / structural molecule activity / virion attachment to host cell / RNA binding / ATP binding
Similarity search - Function
Viral polyprotein, parechovirus P3B / Parechovirus Genome-linked protein / Viral polyprotein, parechovirus P3A / Picornaviridae P3A protein / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Jelly Rolls - #20 / Picornavirales 3C/3C-like protease domain / Picornavirales 3C/3C-like protease domain profile. / Peptidase C3A/C3B, picornaviral / 3C cysteine protease (picornain 3C) ...Viral polyprotein, parechovirus P3B / Parechovirus Genome-linked protein / Viral polyprotein, parechovirus P3A / Picornaviridae P3A protein / Helicase/polymerase/peptidase polyprotein, Calicivirus-type / Jelly Rolls - #20 / 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 / Jelly Rolls / RNA-directed RNA polymerase, catalytic domain / RdRp of positive ssRNA viruses catalytic domain profile. / Peptidase S1, PA clan, chymotrypsin-like fold / DNA/RNA polymerase superfamily / Peptidase S1, PA clan / Sandwich / P-loop containing nucleoside triphosphate hydrolase / Mainly Beta
Similarity search - Domain/homology
RNA / Capsid protein VP0 / Capsid protein VP0
Similarity search - Component
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.
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
Revision 2.1Jul 29, 2020Group: Data collection / Category: em_imaging_optics
Item: _em_imaging_optics.phase_plate / _em_imaging_optics.sph_aberration_corrector

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Structure visualization

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  • Superimposition on EM map
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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 / Source method: isolated from a natural source / Details: RNA / 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 / Source method: isolated from a natural source / Details: polypeptide chain / Source: (natural) Human parechovirus 3 / Cell line: HT29 / Organ: colon adenocarcinoma / References: UniProt: Q8BES5, UniProt: A0A291FGQ4*PLUS

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Antibody , 2 types, 2 molecules HL

#5: Antibody AT12-015 antibody variable heavy


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


Mass: 12339.752 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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 / 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 AT12-015COMPLEXall0MULTIPLE SOURCES
2Human parechovirus type 3VIRUS#1-#41NATURAL
3fabs from AT12-015Semiconductor fabrication plantCOMPLEX#5-#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 / 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) / Num. of grids imaged: 2 / Num. of real images: 6541
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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