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- PDB-7nft: Fujian capbinding domain in complex with Nb8208 -

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Basic information

Entry
Database: PDB / ID: 7nft
TitleFujian capbinding domain in complex with Nb8208
Components
  • Nb8208
  • 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
Polymerase basic protein 2
Similarity search - Component
Biological speciesInfluenza A virus
Homo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.14 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: Nb8208
B: Polymerase basic protein 2
D: Nb8208
hetero molecules


Theoretical massNumber of molelcules
Total (without water)98,69114
Polymers97,7314
Non-polymers96110
Water00
1
A: Polymerase basic protein 2
C: Nb8208
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,3467
Polymers48,8652
Non-polymers4805
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Polymerase basic protein 2
D: Nb8208
hetero molecules


Theoretical massNumber of molelcules
Total (without water)49,3467
Polymers48,8652
Non-polymers4805
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)128.195, 128.195, 70.266
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number168
Space group name H-MP6
Space group name HallP6
Symmetry operation#1: x,y,z
#2: x-y,x,z
#3: y,-x+y,z
#4: -y,x-y,z
#5: -x+y,-x,z
#6: -x,-y,z
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 324 through 482 or resid 501 through 701))
d_2ens_1chain "B"
d_1ens_2chain "C"
d_2ens_2chain "D"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1SERLYSA3 - 161
d_12ens_1SO4SO4B
d_13ens_1SO4SO4C
d_14ens_1SO4SO4D
d_21ens_1SERLYSH1 - 159
d_22ens_1SO4SO4I
d_23ens_1SO4SO4J
d_24ens_1SO4SO4K
d_11ens_2GLNVALE1 - 120
d_12ens_2SO4SO4F
d_13ens_2SO4SO4G
d_21ens_2GLNVALL1 - 120
d_22ens_2SO4SO4M
d_23ens_2SO4SO4N

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(0.667044656877, 0.744951798716, -0.0099117769175), (0.744811857252, -0.667114052521, -0.0146334625485), (-0.0175135099141, 0.00237876402998, -0.999843797026)52.2781953714, -116.19592844, -4.36363424076
2given(0.664736877731, 0.747045235368, 0.00694979837713), (0.747067518386, -0.6647474149, -0.000998676740968), (0.00387380380426, 0.00585582588554, -0.99997535117)52.2673107476, -116.236368007, -5.0920019229

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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 Nb8208


Mass: 14616.222 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Production host: Influenza A virus (A/duck/Fujian/13/2002(H5N1))
#3: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: SO4
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: 2.12 Å3/Da / Density % sol: 41.96 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop
Details: 20% w/v PEG3350, 1 M (NH4)2SO4, 0.1M BIS-TRIS propane pH 5.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 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: 0.9763 Å / Relative weight: 1
ReflectionResolution: 3.14→70.27 Å / Num. obs: 10820 / % possible obs: 92.94 % / Redundancy: 2 % / Biso Wilson estimate: 70.21 Å2 / CC1/2: 0.989 / Net I/σ(I): 9.85
Reflection shellResolution: 3.144→3.256 Å / Num. unique obs: 377 / CC1/2: 0.595

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Processing

Software
NameVersionClassification
PHENIX1.19.1_4122refinement
XDSdata reduction
Aimlessdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6S5V

6s5v


Resolution: 3.14→70.27 Å / SU ML: 0.4419 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 30.2421
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2914 582 5.38 %
Rwork0.2438 10234 -
obs0.2463 10816 92.95 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 71.9 Å2
Refinement stepCycle: LAST / Resolution: 3.14→70.27 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4387 0 50 0 4437
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00214500
X-RAY DIFFRACTIONf_angle_d0.49786056
X-RAY DIFFRACTIONf_chiral_restr0.0412658
X-RAY DIFFRACTIONf_plane_restr0.0023775
X-RAY DIFFRACTIONf_dihedral_angle_d3.9045616
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AX-RAY DIFFRACTIONTorsion NCS0.681474972315
ens_2d_2CX-RAY DIFFRACTIONTorsion NCS0.666248394319
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.14-3.460.32381250.31221952X-RAY DIFFRACTION71.87
3.46-3.960.33191350.27922746X-RAY DIFFRACTION100
3.96-4.990.24861680.22772726X-RAY DIFFRACTION100
4.99-70.270.30071540.21822810X-RAY DIFFRACTION99.76
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
14.076573763341.48040980437-0.3562193824275.66497496844-0.5685689600931.595325117820.360398440631-0.379212131396-0.7356755677190.798267194193-0.214782764213-0.8053272298330.1550747110920.303159173518-0.06969750189910.377652068453-0.0137177458113-0.03740731202280.6279687494620.02626145181710.54324824771251.7971584869-59.665304914225.4256663313
20.02091597031011.31049355038-0.6803106930697.75492460682-1.142090665020.9254783624770.1657070680740.0389671558196-0.0360480703286-0.214351408409-0.300233146708-0.1090222425740.03054994134610.2778817204830.08459671258160.365064277027-0.0100372047866-0.05415993852620.5626202244870.006887823846710.40727863791545.8021452472-54.131990573916.1925009469
34.037485563780.184686982753-0.1739966701643.79072959747-0.1635807213316.18190076464-1.0832718383-0.3402909135461.427060878870.2800178898631.07275634606-0.02996100282150.1320649757780.6630279817090.03327818839590.417426844726-0.0639196665680.04361058500220.550351358104-0.0160265550110.50411154516726.7538614015-26.0809735893-1.67999823916
48.4935155238-0.979228991893-2.883589175284.43902351411-0.300308801272.66473826325-0.2633169234840.64776647549-1.30038482420.144961502921-0.747551587860.3312829693150.563639191227-0.06031229002170.9442799503220.448195057284-0.0421822405722-0.1501371355390.5594183603280.02340847053650.43562266330135.2466783122-32.34083513950.041122795004
56.58292424232-2.16000219284-4.14316106154.738039916410.9282092052272.798021888191.612207964921.626961368361.20714678277-0.269958159104-1.144446461830.2239208578930.274888554768-0.353137431746-0.5535614672190.578124621996-0.0585704331157-0.05990823117140.394295667120.1505869930290.76595649802533.2669350813-22.84138295179.7767995038
66.35950297557-0.328225084786-0.03985678745956.056680929970.8568793193013.81304436121-0.140949880289-0.657283301437-0.2156301212651.28721700325-0.00838506261717-0.5154911068670.7899249464860.09822989920720.4679373894870.590955990874-0.03243927214910.08299809898180.5190674932150.134105723890.38910196665530.1007967728-36.24865627779.47774376582
72.58513625183-0.6610274736241.753563675923.25005839701-0.9134063841351.41170686215-0.507475562586-0.2364880737240.191593216095-0.2027076471520.1567725366770.0700178340493-0.6053613221650.3194111377150.381507429930.513973444708-0.1376941286710.001977984342830.473527387369-0.07218289985490.60869747780832.0026968695-27.6635663824.16165364951
86.01904576246-0.8062967082893.382643029424.882703960950.9071041428342.23192782014-0.711404527672-0.5119796366561.20826811126-0.317475591080.09494453911760.198274152465-0.8066866747520.5192052066680.5374308530630.670837912236-0.139594596689-0.04332423973940.598311625395-0.1386183190990.50014537598637.3599438421-37.3078783882-32.0075601483
96.51797265436-1.466960045410.8305181337898.2858072711-0.8918405611990.1466148165830.177393155572-0.6102965407640.534535811308-0.60595518097-0.414995829265-0.64459091459-0.4705601140860.2275996649630.0782274450080.587452891844-0.06785442356730.06015919828050.641012874413-0.0159934339350.34523665045750.0996958772-42.5693225602-27.465504778
102.13121912105-2.54273869245-1.176600783138.57532705527-0.2253181749256.2084430226-0.321104568093-0.1247848172350.297046110143-0.4794163841310.06541589338111.17804146368-0.692060737956-0.910798239775-0.08701825068640.535402592997-0.1270725899070.005052966831920.556934914804-0.1390205571310.8022297621245.4742226391-43.3066739614-20.2119995954
115.18362217317-0.330196752858-0.141886729726.513191691722.936589015693.39889839558-0.0981544018157-0.271765012873-0.4120930548630.2042600669060.05688492539251.05574213904-0.0118366507659-0.411101217394-0.1874291644060.463018480049-0.030331762326-0.08134381428480.5530428981370.1045385852010.44114763265230.1069743151-37.4443594307-21.4138318422
124.34084091931-4.294786683331.996365763814.13864664036-2.284210860991.77044412071-0.716204032391-0.648713879840.210403916411.32532182230.8420699238320.296614913919-0.0839683941828-0.651795463203-0.1597327242090.8359368737550.01336530152090.07835899368730.646735953990.05541014869490.44541295039836.3221269768-43.653200241-19.4704952355
132.295695414322.369654996820.128838390383.43914149801-1.345344432532.33855842622-0.19060260220.02444347939540.226296951882-0.2497814633810.123925174067-1.26711582395-0.1641248466271.02419378380.0949122068890.51884506268-0.1788736188070.03118415869050.737430471915-0.1471144142950.65538853648751.8319597908-56.5522723163-22.5289478561
148.33760475038-4.02095779672-1.359162440678.49362958583-0.7420254436366.615159328720.444873945132.10570845813-0.144982884611-0.649668196944-1.09534377530.8659910179080.8735011336520.3781426143470.601050188930.411404957137-0.0230196628939-0.09398345550610.4755850975010.0346778548360.65445662542751.6110304884-55.3727582796-22.5326821333
157.262144244491.12887733015-1.008030883827.63762497935-1.184972630038.53367262039-0.295788833285-0.7211176276010.4703310688480.16824870503-0.3567337697240.3742122036480.03336710585610.6516339512150.7386009757370.496988892135-0.0547910158324-0.01195873542060.5772764810240.07049584353820.57724489043151.1289116511-72.5725911046-4.59694341575
169.48101272350.946829204722.839230698133.50602043185-2.051701899336.382796769770.02206604599142.57423200425-1.15090115999-0.160225241274-0.874578361914-1.1812573952-0.127321626881.463358020790.3917165155320.437203868348-0.03090494825770.07441913184650.8049805080680.04033456552770.67067230591457.5348389639-76.1739975169-14.5444836525
174.669662219241.165978703591.454299229195.45541742725-1.365657877172.45015357685-0.290806641730.3767650839860.142753977817-0.7537490881630.222263383005-0.312339298501-0.1231149354820.4090387598680.0460681416920.437377973749-0.06880854704020.03779533644620.453502527585-0.02011754484550.37584188423750.5075619358-72.5821788491-11.0269602755
Refinement TLS group

Refine-ID: X-RAY DIFFRACTION

IDRefine TLS-IDSelection detailsAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11chain 'A' and (resid 322 through 379 )AA322 - 3791 - 58
22chain 'A' and (resid 380 through 482 )AA380 - 48259 - 161
33chain 'C' and (resid 1 through 17 )CE1 - 171 - 17
44chain 'C' and (resid 18 through 39 )CE18 - 3918 - 39
55chain 'C' and (resid 40 through 51 )CE40 - 5140 - 51
66chain 'C' and (resid 52 through 73 )CE52 - 7352 - 73
77chain 'C' and (resid 74 through 120 )CE74 - 12074 - 120
88chain 'B' and (resid 324 through 358 )BH324 - 3581 - 35
99chain 'B' and (resid 359 through 390 )BH359 - 39036 - 67
1010chain 'B' and (resid 391 through 405 )BH391 - 40568 - 82
1111chain 'B' and (resid 406 through 429 )BH406 - 42983 - 106
1212chain 'B' and (resid 430 through 449 )BH430 - 449107 - 126
1313chain 'B' and (resid 450 through 468 )BH450 - 468127 - 145
1414chain 'B' and (resid 469 through 482 )BH469 - 482146 - 159
1515chain 'D' and (resid 1 through 39 )DL1 - 391 - 39
1616chain 'D' and (resid 40 through 51 )DL40 - 5140 - 51
1717chain 'D' and (resid 52 through 120 )DL52 - 12052 - 120

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