2020-11-18RELPDBePDBePDBe2019-03-072019-03-202019-09-042020-11-18Wellcome Trust200835/Z/16/ZUnited KingdomMedical Research Council (United Kingdom)MR/K000241/1United KingdomMedical Research Council (United Kingdom)MR/R009945/1United KingdomInfluenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb8205Carrique LKeown JRFan HFodor EGrimes JMFan HWalker APCarrique LKeown JRSerna Martin IKaria DSharps JHengrung NPardon ESteyaert JGrimes JMFodor EStructures of influenza A virus RNA polymerase offer insight into viral genome replication.NatureUK573287290201931485076doi:10.1038/s41586-019-1530-71476-46870006NATUASEMD-4661other EM volumeEMD-4666associated EM volumeInfluenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb82056qxeFULLOVERLAPInfluenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb8205Influenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb8205012345Sample was treated with 0.001% glutaraldehyde for 20 min on ice prior quenching with 100 mM Tris-HCl pH 7.5 and gel filtration.0.25Influenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb820511234Influenza A virus (A/nt/60/1968(H3N2))Spodoptera frugiperdaInfluenza A virus (A/NT/60/1968) polymerase dimer of hetermotrimer in complex with 3'5' cRNA promoter and Nb820515Influenza A virus (A/nt/60/1968(H3N2))synthetic constructNb8205 Influenza A virus (A/nt/60/1968(H3N2))0.0148353752Spodoptera frugiperdaLEVOQVQLQESGGGMVQPGGSLRLSCLASGFTFSNYAMTWVRQAPGKGPEWVSMVSNNGADTTYTDSVKGRFTISRDNAKNTLY
LRMNNVKPEDSAVYYCAKRRYGGIWTGQPTDYDYLGQGTVTVSSHHHHHHEPEAPolymerase acidic protein Influenza A virus (A/nt/60/1968(H3N2))0.0831007972Spodoptera frugiperdaLEVOMEDFVRQCFNPMIVELAEKAMKEYGEDLKIETNKFAAICTHLEVCFMYSDFHFINEQGESIVVELDDPNALLKHRFEIIE
GRDRTMAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKSENTHIHIFSFTGEEMATKAD
YTLDEESRARIKTRLFTIRQEMANRGLWDSFRQSERGEETIEERFEITGTMRRLADQSLPPNFSCLENFRAYVDGFEPNG
YIEGKLSQMSKEVNAKIEPFLKTTPRPIRLPDGPPCFQRSKFLLMDALKLSIEDPSHEGEGIPLYDAIKCMRTFFGWKEP
YIVKPHEKGINPNYLLSWKQVLAELQDIENEEKIPRTKNMKKTSQLKWALGENMAPEKVDFDNCRDVSDLKQYDSDEPEL
RSLSSWIQNEFNKACELTDSTWIELDEIGEDVAPIEYIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDF
QLIPMISKCRTKEGRRKTNLYGFIIKGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRSAIGQMSRP
MFLYVRTNGTSKIKMKWGMEMRRCLLQSLQQIESMIEAESSVKEKDMTKEFFENKSETWPIGESPKGVEDGSIGKVCRTL
LAKSVFNSLYASPQLEGFSAESRKLLLVVQALRDNLEPGTFDLEGLYEAIEECLINDPWVLLNASWFNSFLTHALR3.1.-.-RNA-directed RNA polymerase catalytic subunit Influenza A virus (A/nt/60/1968(H3N2))0.0865240862Spodoptera frugiperdaLEVOMDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTETGAPQLNPIDGPLPEDNEPS
GYAQTDCVLEAMAFLEESHPGIFENSCLETMEVVQQTRVDRLTQGRQTYDWTLNRNQPAATALANTIEVFRSNGLTANES
GRLIDFLKDVMESMDKEEMEITTHFQRKRRVRDNMTKKMVTQRTIGKKKQRVNKRSYLIRALTLNTMTKDAERGKLKRRA
IATPGMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKAKLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRMFLA
MITYITKNQPEWFRNVLSIAPIMFSNKMARLGKGYMFESKSMKLRTQIPAEMLASIDLKYFNESTRKKIEKIRPLLIDGT
ASLSPGMMMGMFNMLSTVLGVSILNLGQKRYTKTTYWWDGLQSSDDFALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKK
KSYINRTGTFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMINNDLGPATAQMALQLFIKDYRYTYR
CHRGDTQIQTRRSFELKKLWEQTRSKAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCNPLNPFVSHKEIESV
NNAVVMPAHGPAKSMEYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFFPSSSYRRPVGISSMVEAMVS
RARIDARIDFESGRIKKEEFAEIMKICSTIEELRRQK2.7.7.48Polymerase basic protein 2 Influenza A virus (A/nt/60/1968(H3N2))0.0861633912Spodoptera frugiperdaLEVOMERIKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKRITEMVPERNEQGQTLWSK
MSDAGSDRVMVSPLAVTWWNRNGPMTSTVHYPKVYKTYFEKVERLKHGTFGPVHFRNQVKIRRRVDINPGHADLSAKEAQ
DVIMEVVFPNEVGARILTSESQLTITKEKKEELQDCKISPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCW
EQMYTPGGEVRNDDVDQSLIIAARNIVRRAAVSADPLASLLEMCHSTQIGGTRMVDILRQNPTEEQAVDICKAAMGLRIS
SSFSFGGFTFKRTSGSSIKREEELLTGNLQTLKIRVHDGYEEFTMVGKRATAILRKATRRLVQLIVSGRDEQSVAEAIIV
AMVFSQEDCMIKAVRGDLNFVNRANQRLNPMHQLLRHFQKDAKVLFQNWGIEHIDNVMGMIGVLPDMTPSTEMSMRGIRV
SKMGVDEYSSTERVVVSIDRFLRVRDQRGNVLLSPEEVSETQGTEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETV
KIQWSQNPTMLYNKMEFEPFQSLVPKAIRGQYSGFVRTLFQQMRDVLGTFDTTQIIKLLPFAAAPPKQSRMQFSSLTVNV
RGSGMRILVRGNSPAFNYNKTTKRLTILGKDAGTLIEDPDEGTSGVESAVLRGFLILGKEDRRYGPALSINELSNLAKGE
KANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAINRNA (5'-R(P*AP*GP*CP*AP*AP*AP*AP*GP*CP*AP*GP*G)-3')Influenza A virus (A/nt/60/1968(H3N2))0.0049010972(P)AGCAAAAGCAGGCCsingleParticleparticle0.357.520.0HEPES150.0NaClSample 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.ETHANE100298FEI VITROBOT MARK IVblot for 3.5 sec before plunging. FEI TITAN KRIOSFLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN3002.70.50.7130000.0FEI TITAN KRIOS AUTOGRID HOLDERNITROGENVOLTA PHASE PLATEGATAN K2 SUMMIT (4k x 4k)COUNTING124566.01.251406945template picking in cryosparc v2.5Gctf1.18Ab initio model was generated by cryoSPARCC14.15FSC 0.143 CUT-OFFRELION327861MAXIMUM LIKELIHOODRELION3RELION 3MAXIMUM LIKELIHOODRELION3RELION36QX8A6QX8B6QX8C6QX8D6QPGM6QPGORIGID BODY FITRigid body fit onlyREAL95