[English] 日本語
Yorodumi
- PDB-6qx8: Influenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 6qx8
TitleInfluenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer in complex with 5' cRNA promoter
Components
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • RNA (5'-R(P*AP*GP*CP*AP*AP*AP*AP*GP*CP*AP*GP*A)-3')
  • RNA-directed RNA polymerase catalytic subunit
KeywordsRNA BINDING PROTEIN / Influenza A / RNA polymerase / Influenza polymerase / Influenza dimer / RDRP
Function / homology
Function and homology information


cap snatching / suppression by virus of host RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / viral transcription / suppression by virus of host MAVS activity / virion / RNA-directed RNA polymerase / viral RNA genome replication / endonuclease activity ...cap snatching / suppression by virus of host RNA polymerase II activity / host cell mitochondrion / 7-methylguanosine mRNA capping / viral transcription / suppression by virus of host MAVS activity / virion / RNA-directed RNA polymerase / viral RNA genome replication / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity / host cell cytoplasm / Hydrolases, Acting on ester bonds / transcription, DNA-templated / host cell nucleus / nucleotide binding / RNA binding / metal ion binding
Influenza RNA-dependent RNA polymerase subunit PB2 / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PB1 / PA/PA-X superfamily / Polymerase acidic protein / PB2, C-terminal / Influenza RNA-dependent RNA polymerase subunit PB1 / Influenza RNA-dependent RNA polymerase subunit PB2 / Influenza RNA-dependent RNA polymerase subunit PA / RNA-directed RNA polymerase, negative-strand RNA virus
Polymerase basic protein 2 / RNA-directed RNA polymerase catalytic subunit / Polymerase acidic protein
Biological speciesInfluenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.07 Å
AuthorsCarrique, L. / Keown, J.R. / Fan, H. / Fodor, E. / Grimes, J.M.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (United Kingdom)MR/R009945/1 United Kingdom
Medical Research Council (United Kingdom)MR/K000241/1 United Kingdom
CitationJournal: Nature / Year: 2019
Title: Structures of influenza A virus RNA polymerase offer insight into viral genome replication.
Authors: Haitian Fan / Alexander P Walker / Loïc Carrique / Jeremy R Keown / Itziar Serna Martin / Dimple Karia / Jane Sharps / Narin Hengrung / Els Pardon / Jan Steyaert / Jonathan M Grimes / Ervin Fodor /
Abstract: Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans. Influenza A viruses contain a segmented ...Influenza A viruses are responsible for seasonal epidemics, and pandemics can arise from the transmission of novel zoonotic influenza A viruses to humans. Influenza A viruses contain a segmented negative-sense RNA genome, which is transcribed and replicated by the viral-RNA-dependent RNA polymerase (FluPol) composed of PB1, PB2 and PA subunits. Although the high-resolution crystal structure of FluPol of bat influenza A virus has previously been reported, there are no complete structures available for human and avian FluPol. Furthermore, the molecular mechanisms of genomic viral RNA (vRNA) replication-which proceeds through a complementary RNA (cRNA) replicative intermediate, and requires oligomerization of the polymerase-remain largely unknown. Here, using crystallography and cryo-electron microscopy, we determine the structures of FluPol from human influenza A/NT/60/1968 (H3N2) and avian influenza A/duck/Fujian/01/2002 (H5N1) viruses at a resolution of 3.0-4.3 Å, in the presence or absence of a cRNA or vRNA template. In solution, FluPol forms dimers of heterotrimers through the C-terminal domain of the PA subunit, the thumb subdomain of PB1 and the N1 subdomain of PB2. The cryo-electron microscopy structure of monomeric FluPol bound to the cRNA template reveals a binding site for the 3' cRNA at the dimer interface. We use a combination of cell-based and in vitro assays to show that the interface of the FluPol dimer is required for vRNA synthesis during replication of the viral genome. We also show that a nanobody (a single-domain antibody) that interferes with FluPol dimerization inhibits the synthesis of vRNA and, consequently, inhibits virus replication in infected cells. Our study provides high-resolution structures of medically relevant FluPol, as well as insights into the replication mechanisms of the viral RNA genome. In addition, our work identifies sites in FluPol that could be targeted in the development of antiviral drugs.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMar 7, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-4663
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Polymerase acidic protein
B: RNA-directed RNA polymerase catalytic subunit
C: Polymerase basic protein 2
D: RNA (5'-R(P*AP*GP*CP*AP*AP*AP*AP*GP*CP*AP*GP*A)-3')
E: Polymerase acidic protein
F: RNA-directed RNA polymerase catalytic subunit
G: Polymerase basic protein 2
H: RNA (5'-R(P*AP*GP*CP*AP*AP*AP*AP*GP*CP*AP*GP*A)-3')


Theoretical massNumber of molelcules
Total (without water)521,3798
Polymers521,3798
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

-
Components

#1: Protein/peptide Polymerase acidic protein / RNA-directed RNA polymerase subunit P2


Mass: 83100.797 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Gene: PA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P03434, Hydrolases, Acting on ester bonds
#2: Protein/peptide RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 1 / PB1 / RNA-directed RNA polymerase subunit P1


Mass: 86524.086 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03432, RNA-directed RNA polymerase
#3: Protein/peptide Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 86163.391 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (strain A/Northern Territory/60/1968 H3N2)
Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P03429
#4: RNA chain RNA (5'-R(P*AP*GP*CP*AP*AP*AP*AP*GP*CP*AP*GP*A)-3')


Mass: 4901.097 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Influenza A virus (A/nt/60/1968(H3N2))

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

-
Sample preparation

Component

Name: Influenza A virus (A/NT/60/1968) polymerase dimer of heterotrimer in complex with 5' cRNA promoter / Source: RECOMBINANT / Type: COMPLEX

IDDetailsEntity IDParent-ID
1Sample was treated with 0.001% glutaraldehyde for 20 min on ice prior quenching with 100 mM Tris-HCl pH 7.5 and gel filtration.1, 2, 3, 40
21, 2, 31
341
Molecular weightValue: 0.25 MDa / Experimental value: NO
Source (natural)

Ncbi tax-ID: 384505 / Organism: Influenza A virus (A/nt/60/1968(H3N2))

IDEntity assembly-ID
22
33
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Details: Sample was purified in 20 mM HEPES, pH 7.5, 150 mM NaCl with Tween 20 added to a final concentration 0f 0.05% prior to plunging grids.
Buffer component

Buffer-ID: 1

IDConc.NameFormula
120 mMHEPES
2150 mMNaClSodium chloride
SpecimenConc.: 0.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: blot for 3.5 sec before plunging

-
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: 130000 X / Nominal defocus max: 700 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5 sec. / Electron dose: 1.32 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1178
EM imaging opticsPhase plate: Volta phase plate
Image scansMovie frames/image: 25

-
Processing

Software
NameVersionClassification
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.18CTF correction
7UCSF Chimera1.13model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 56070
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 36913 / Symmetry type: POINT
Atomic model buildingB value: 100 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6QNW
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.006539068
f_angle_d0.97670564
f_chiral_restr0.04742992
f_plane_restr0.00545688
f_dihedral_angle_d15.505215728

+
About Yorodumi

-
News

-
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force. (see PDBe EMDB page)
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.: Q: What is "EMD"? / ID/Accession-code notation in Yorodumi/EM Navigator

External links: EMDB at PDBe / Contact to PDBj

-
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software). Now, EM Navigator and Yorodumi are based on the updated data.

External links: wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

+
Jun 16, 2017. Omokage search with filter

Omokage search with filter

  • Result of Omokage search can be filtered by keywords and the database types

Related info.: Omokage search

+
Sep 15, 2016. EM Navigator & Yorodumi renewed

EM Navigator & Yorodumi renewed

  • New versions of EM Navigator and Yorodumi started

Related info.: Changes in new EM Navigator and Yorodumi

+
Aug 31, 2016. New EM Navigator & Yorodumi

New EM Navigator & Yorodumi

  • In 15th Sep 2016, the development versions of EM Navigator and Yorodumi will replace the official versions.
  • Current version will continue as 'legacy version' for some time.

Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator / Yorodumi

Read more

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.

Related info.: EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more