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- PDB-6qwl: Influenza B virus (B/Panama/45) polymerase Hetermotrimer in compl... -

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Basic information

Entry
Database: PDB / ID: 6qwl
TitleInfluenza B virus (B/Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter
Components
  • 3' cRNA
  • 5' cRNA
  • Polymerase acidic protein
  • Polymerase basic protein 2
  • 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 / 7-methylguanosine mRNA capping / viral transcription / virion / RNA-directed RNA polymerase / viral RNA genome replication / endonuclease activity / RNA-directed 5'-3' RNA polymerase activity / host cell cytoplasm ...cap snatching / suppression by virus of host RNA polymerase II activity / 7-methylguanosine mRNA capping / viral transcription / 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 PA / RdRp of negative ssRNA viruses with segmented genomes catalytic domain profile. / Influenza RNA-dependent RNA polymerase subunit PB2 / Influenza RNA-dependent RNA polymerase subunit PA / Influenza RNA-dependent RNA polymerase subunit PB1 / PA/PA-X superfamily / Polymerase acidic protein / PB2, C-terminal / RNA-directed RNA polymerase, negative-strand RNA virus / Influenza RNA-dependent RNA polymerase subunit PB2 / Influenza RNA-dependent RNA polymerase subunit PB1
RNA-directed RNA polymerase catalytic subunit / Polymerase basic protein 2 / Polymerase acidic protein
Biological speciesInfluenza B virus
Influenza A virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsKeown, J.R. / Carrique, L. / 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 5, 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

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4660
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
E: Polymerase acidic protein
K: RNA-directed RNA polymerase catalytic subunit
Q: Polymerase basic protein 2
T: 3' cRNA
W: 5' cRNA


Theoretical massNumber of molelcules
Total (without water)265,8525
Polymers265,8525
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: scanning transmission electron microscopy
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 83161.055 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus (strain B/Panama/45/1990)
Gene: PA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: O36432, 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: 84378.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus (strain B/Panama/45/1990)
Gene: PB1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O36430, RNA-directed RNA polymerase
#3: Protein/peptide Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 89097.453 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza B virus (strain B/Panama/45/1990)
Gene: PB2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: O36431
#4: RNA chain 3' cRNA


Mass: 4683.753 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus
#5: RNA chain 5' cRNA


Mass: 4531.827 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Influenza A virus

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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

Details: Sample was treated with 0.001% glutaraldehyde for 20 min on ice prior quenching with 100 mM Tris-HCl pH 7.5 and gel filtration. / Name: Influenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter / Source: RECOMBINANT / Type: COMPLEX

IDEntity IDParent-ID
11, 2, 3, 4, 50
21, 2, 31
34, 51
Molecular weightValue: 0.25 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Influenza B virus (B/Panama/45/1990)408929
33Influenza A virus11320
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-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: blot for 3.5 sec 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: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.38 sec. / Electron dose: 1.1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4711
Image scansMovie frames/image: 28 / Used frames/image: 1-28

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.18CTF correction
7UCSF Chimera1.13model fitting
9cryoSPARC2.5initial Euler assignment
10cryoSPARC2.5final Euler assignment
11cryoSPARC2.5classification
12cryoSPARC2.53D reconstruction
13PHENIX1.14model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1012085 / Details: template picking in cryosparc v2.5
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1012085 / Symmetry type: POINT
Atomic model buildingB value: 57 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 5EPI
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.007710669
f_angle_d0.995614525
f_chiral_restr0.06191625
f_plane_restr0.00771768
f_dihedral_angle_d7.6666410

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