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- PDB-7nfq: Fujian capmidlink domain in complex with Nb8193 -

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Basic information

Entry
Database: PDB / ID: 7nfq
TitleFujian capmidlink domain in complex with Nb8193
Components
  • Nb8193
  • Polymerase basic protein 2
KeywordsVIRAL PROTEIN / Influenza polymerase / cap-binding domain / nanobody
Function / homology
Function and homology information


cap snatching / symbiont-mediated suppression of host mRNA transcription via inhibition of RNA polymerase II activity / 7-methylguanosine mRNA capping / host cell mitochondrion / virion component / symbiont-mediated suppression of host gene expression / viral RNA genome replication / RNA-directed RNA polymerase activity / DNA-templated transcription / host cell nucleus / RNA binding
Similarity search - Function
Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain / : / Influenza RNA polymerase PB2 C-terminal domain ...Influenza RNA-dependent RNA polymerase subunit PB2 / PB2, C-terminal / : / : / Influenza RNA polymerase PB2 N-terminal region / Influenza RNA polymerase PB2 second domain / : / Influenza RNA polymerase PB2 middle domain / : / Influenza RNA polymerase PB2 C-terminal domain / : / Influenza RNA polymerase PB2 6th domain / : / Influenza RNA polymerase PB2 CAP binding domain / : / Influenza RNA polymerase PB2 helical domain / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
DI(HYDROXYETHYL)ETHER / Polymerase basic protein 2
Similarity search - Component
Biological speciesInfluenza A virus
Camelidae mixed library (mammal)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.68 Å
AuthorsKeown, J.R. / Grimes, J.M. / Fodor, E.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust200835/Z/16/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/R009945/1 United Kingdom
CitationJournal: Nat Commun / Year: 2022
Title: Mapping inhibitory sites on the RNA polymerase of the 1918 pandemic influenza virus using nanobodies.
Authors: Jeremy R Keown / Zihan Zhu / Loïc Carrique / Haitian Fan / Alexander P Walker / Itziar Serna Martin / Els Pardon / Jan Steyaert / Ervin Fodor / Jonathan M Grimes /
Abstract: Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA- ...Influenza A viruses cause seasonal epidemics and global pandemics, representing a considerable burden to healthcare systems. Central to the replication cycle of influenza viruses is the viral RNA-dependent RNA polymerase which transcribes and replicates the viral RNA genome. The polymerase undergoes conformational rearrangements and interacts with viral and host proteins to perform these functions. Here we determine the structure of the 1918 influenza virus polymerase in transcriptase and replicase conformations using cryo-electron microscopy (cryo-EM). We then structurally and functionally characterise the binding of single-domain nanobodies to the polymerase of the 1918 pandemic influenza virus. Combining these functional and structural data we identify five sites on the polymerase which are sensitive to inhibition by nanobodies. We propose that the binding of nanobodies at these sites either prevents the polymerase from assuming particular functional conformations or interactions with viral or host factors. The polymerase is highly conserved across the influenza A subtypes, suggesting these sites as effective targets for potential influenza antiviral development.
History
DepositionFeb 7, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 1, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 9, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.3Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polymerase basic protein 2
C: Nb8193
B: Polymerase basic protein 2
D: Nb8193
hetero molecules


Theoretical massNumber of molelcules
Total (without water)97,03210
Polymers96,4654
Non-polymers5676
Water8,161453
1
A: Polymerase basic protein 2
C: Nb8193
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,3253
Polymers48,2332
Non-polymers921
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Polymerase basic protein 2
D: Nb8193
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,7077
Polymers48,2332
Non-polymers4745
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)42.583, 106.543, 98.405
Angle α, β, γ (deg.)90.000, 98.007, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein Polymerase basic protein 2 / RNA-directed RNA polymerase subunit P3


Mass: 34249.168 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Influenza A virus (A/duck/Fujian/13/2002(H5N1))
Gene: PB2 / Production host: Escherichia coli (E. coli) / References: UniProt: Q6E3N3
#2: Antibody Nb8193


Mass: 13983.466 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelidae mixed library (mammal) / Production host: Escherichia coli (E. coli)
#3: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C3H8O3
#4: Chemical ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H10O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 453 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.29 Å3/Da / Density % sol: 46.32 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / Details: 20% w/v PEG3350 and 0.2 M (NH4)2 citrate

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9762 Å
DetectorType: DECTRIS EIGER2 X 16M / Detector: PIXEL / Date: May 14, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9762 Å / Relative weight: 1
ReflectionResolution: 1.68→44.31 Å / Num. obs: 69149 / % possible obs: 70.31 % / Redundancy: 2 % / Biso Wilson estimate: 28.16 Å2 / CC1/2: 1 / Net I/σ(I): 23.17
Reflection shellResolution: 1.68→1.743 Å / Num. unique obs: 767 / CC1/2: 0.292

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Processing

Software
NameVersionClassification
PHENIX1.18rc2_3794refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6S5V

6s5v


Resolution: 1.68→44.31 Å / SU ML: 0.1727 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 28.9662
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2054 3442 4.98 %
Rwork0.1673 65692 -
obs0.1693 69134 70.32 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 35.39 Å2
Refinement stepCycle: LAST / Resolution: 1.68→44.31 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms6175 0 37 453 6665
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0136326
X-RAY DIFFRACTIONf_angle_d1.20928522
X-RAY DIFFRACTIONf_chiral_restr0.0642966
X-RAY DIFFRACTIONf_plane_restr0.00761105
X-RAY DIFFRACTIONf_dihedral_angle_d21.8999895
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.68-1.710.4557130.3249149X-RAY DIFFRACTION4.11
1.71-1.730.4175170.3774331X-RAY DIFFRACTION9.18
1.73-1.760.3049370.3328546X-RAY DIFFRACTION14.86
1.76-1.780.3905510.2964744X-RAY DIFFRACTION20.27
1.78-1.810.324570.2654999X-RAY DIFFRACTION27.2
1.81-1.840.3331670.24331255X-RAY DIFFRACTION33.26
1.84-1.880.2587820.23211446X-RAY DIFFRACTION39.41
1.88-1.910.2672910.22781771X-RAY DIFFRACTION47.36
1.91-1.950.2447950.20182047X-RAY DIFFRACTION54.7
1.95-20.21071220.19792384X-RAY DIFFRACTION64.37
2-2.040.24381420.19422698X-RAY DIFFRACTION71.36
2.04-2.090.20711530.1893013X-RAY DIFFRACTION81.24
2.09-2.150.23711680.18363407X-RAY DIFFRACTION91.2
2.15-2.210.2171690.1743673X-RAY DIFFRACTION98.21
2.21-2.280.21141860.17583736X-RAY DIFFRACTION99.9
2.28-2.370.2581900.17393794X-RAY DIFFRACTION100
2.37-2.460.24541880.17533718X-RAY DIFFRACTION100
2.46-2.570.21532040.18053691X-RAY DIFFRACTION100
2.57-2.710.21811990.17993752X-RAY DIFFRACTION99.97
2.71-2.880.21441830.19053766X-RAY DIFFRACTION99.97
2.88-3.10.23412040.18323739X-RAY DIFFRACTION99.95
3.1-3.410.23551910.17063734X-RAY DIFFRACTION99.97
3.41-3.90.17782010.14843774X-RAY DIFFRACTION99.97
3.9-4.920.14782070.12483731X-RAY DIFFRACTION99.92
4.92-44.310.19822250.16453794X-RAY DIFFRACTION99.68
Refinement TLS params.Method: refined / Origin x: 4.58878721433 Å / Origin y: 10.3509992209 Å / Origin z: 18.7829446686 Å
111213212223313233
T0.210733936277 Å20.00569372118006 Å2-0.00958883075485 Å2-0.197455039602 Å20.00180330601925 Å2--0.276819494776 Å2
L0.0417103759732 °20.0222759489325 °2-0.0986884221022 °2-0.0122726344801 °2-0.109218053678 °2--0.681990908568 °2
S0.000581697943881 Å °-0.0191182496657 Å °0.0100472038526 Å °0.0291711404904 Å °-0.00381424047408 Å °-0.0239827113348 Å °-0.0430753534364 Å °0.0536541944941 Å °0.00464636594937 Å °
Refinement TLS groupSelection details: all

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