2019-12-18RELPDBePDBePDBe2019-03-052019-03-132019-09-042019-12-18Wellcome Trust200835/Z/16/ZUnited KingdomMedical Research Council (United Kingdom)MR/K000241/1United KingdomMedical Research Council (United Kingdom)MR/R009945/1United KingdomInfluenza B virus (B/Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoterKeown JRCarrique LFan 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-4660associated EM volumeInfluenza B virus (B/Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter6qwlFULLOVERLAPInfluenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoterInfluenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter012345Sample 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 B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter1123Sample was treated with 0.001% glutaraldehyde for 20 min on ice prior quenching with 100 mM Tris-HCl pH 7.5 and gel filtration.Influenza B virus (B/Panama/45/1990)Spodoptera frugiperdaInfluenza B (Panama/45) polymerase Hetermotrimer in complex with 3'5' cRNA promoter145Sample was treated with 0.001% glutaraldehyde for 20 min on ice prior quenching with 100 mM Tris-HCl pH 7.5 and gel filtration.Influenza A virussynthetic constructPolymerase acidic proteinInfluenza B virus (strain B/Panama/45/1990)0.0831610551Spodoptera frugiperdaLEVOMDTFITRNFQTTIIQKAKNTMAEFSEDPELQPAMLFNICVHLEVCYVISDMNFLDEEGKSYTALEGQGKEQNLRPQYEVI
EGMPRTIAWMVQRSLAQEHGIETPKYLADLFDYKTKRFIEVGITKGLADDYFWKKKEKLGNSMELMIFSYNQDYSLSNES
SLDEEGKGRVLSRLTELQAELSLKNLWQVLIGEEDVEKGIDFKLGQTISRLRDISVPAGFSNFEGMRSYIDNIDPKGAIE
RNLARMSPLVSATPKKLKWEDLRPIGPHIYNHELPEVPYNAFLLMSDELGLANMTEGKSKKPKTLAKECLEKYSTLRDQT
DPILIMKSEKANENFLWKLWRDCVNTISNEEMSNELQKTNYAKWATGDGLTYQKIMKEVAIDDETMCQEEPKIPNKCRVA
AWVQTEMNLLSTLTSKRALDLPEIGPDVAPVEHVGSERRKYFVNEINYCKASTVMMKYVLFHTSLLNESNASMGKYKVIP
ITNRVVNEKGESFDMLYGLAVKGQSHLRGDTDVVTVVTFEFSSTDPRVDSGKWPKYTVFRIGSLFVSGREKSVYLYCRVN
GTNKIQMKWGMEARRCLLQSMQQMEAIVEQESSIQGYDMTKACFKGDRVNSPKTFSIGTQEGKLVKGSFGKALRVIFTKC
LMHYVFGNAQLEGFSAESRRLLLLIQALKDRKGPWVFDLEGMYSGIEECISNNPWVIQSAYWFNEWLGFEKEGSKVLESV
DEIMDE3.1.-.-RNA-directed RNA polymerase catalytic subunitInfluenza B virus (strain B/Panama/45/1990)0.084378181Spodoptera frugiperdaLEVOMNINPYFLFIDVPIQAAISTTFPYTGVPPYSHGTGTGHTIDTVIRTHEYSNKGKQYVSDVTGCTMVDPTNGPLPEDNEPS
AYAQLDCVLEALDRMDEEHPGLFQAASQNAMEALMVTTVDKLTQGRQTFDWTVCRNQPAATALNTTITSFRLNDLNGADK
GGLVPFCQDIIDSLDKPEMTFFSVKNIKKKLPAKNRKGFLIKRIPMKVKDRITRVEYIKRALSLNTMTKDAERGKLKRRA
IATAGIQIRGFVLVVENLAKNICENLEQSGLPVGGNEKKAKLSNAVAKMLSNCPPGGISMTVTGDNTKWNECLNPRIFLA
MTERITRDSPIWFRDFCSIAPVLFSNKIARLGKGFMITSKTKRLKAQIPCPDLFSIPLERYNEETRAKLKKLKPFFNEEG
TASLSPGMMMGMFNMLSTVLGVAALGIKNIGNKEYLWDGLQSSDDFALFVNAKDEETCMEGINDFYRTCKLLGINMSKKK
SYCNETGMFEFTSMFYRDGFVSNFAMEIPSFGVAGVNESADMAIGMTIIKNNMINNGMGPATAQTAIQLFIADYRYTYKC
HRGDSKVEGKRMKIIKELWENTKGRDGLLVADGGPNIYNLRNLHIPEIVLKYNLMDPEYKGRLLHPQNPFVGHLSIEGIK
EADITPAHGPVKKMDYDAVSGTHSWRTKRNRSILNTDQRNMILEEQCYAKCCNLFEACFNSASYRKPVGQHSMLEAMAHR
LRMDARLDYESGRMSKDDFEKAMAHLGEIGYI2.7.7.48Polymerase basic protein 2Influenza B virus (strain B/Panama/45/1990)0.0890974531Spodoptera frugiperdaLEVOMTLAKIELLKQLLRDNEAKTVLKQTTVDQYNIIRKFNTSRIEKNPSLRMKWAMCSNFPLALTKGDMANRIPLEYKGIQLK
TNAEDIGTKGQMCSIAAVTWWNTYGPIGDTEGFEKVYESFFLRKMRLDNATWGRITFGPVERVRKRVLLNPLTKEMPPDE
ASNVIMEILFPKEAGIPRESTWIHRELIKEKREKLKGTMITPIVLAYMLERELVARRRFLPVAGATSAEFIEMLHCLQGE
NWRQIYHPGGNKLTESRSQSMIVACRKIIRRSIVASNPLELAVEIANKTVIDTEPLKSCLTAIDGGDVACDIIRAALGLK
IRQRQRFGRLELKRISGRGFKNDEEILIGNGTIQKIGIWDGEEEFHVRCGECRGILKKSKMRMEKLLINSAKKEDMKDLI
ILCMVFSQDTRMFQGVRGEINFLNRAGQLLSPMYQLQRYFLNRSNDLFDQWGYEESPKASELHGINELMNASDYTLKGVV
VTKNVIDDFSSTETEKVSITKNLSLIKRTGEVIMGANDVSELESQAQLMITYDTPKMWEMGTTKELVQNTYQWVLKNLVT
LKAQFLLGKEDMFQWDAFEAFESIIPQKMAGQYSGFARAVLKQMRDQEVMKTDQFIKLLPFCFSPPKLRSNGEPYQFLRL
VLKGGGENFIEVRKGSPLFSYNPQTEVLTICGRMMSLKGKIEDEERNRSMGNAVLAGFLVSGKYDPDLGDFKTIEELEKL
KPGEKANILLYQGKPVKVVKRKRYSALSNDISQGIKRQRMTVESMGWALSARENLYFQ3' cRNAInfluenza A virus0.0046837531GGCCUUGUUUCUACU5' cRNAInfluenza A virus0.0045318271AGCAAAAGCAGGCCsingleParticleparticle0.357.520.0HEPES150.0NaClSample 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.C-flat-2/1COPPER200CARBONHOLEYGLOW DISCHARGEAIR101.325ETHANE100298FEI VITROBOT MARK IVblot for 3.5 sec before plunging. FEI TITAN KRIOSFLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN30070.02.71.32.5130000.0FEI TITAN KRIOS AUTOGRID HOLDERNITROGENGATAN K2 SUMMIT (4k x 4k)COUNTING1-28147114.381.111012085template picking in cryosparc v2.5Gctf1.18Ab initio model was generated by cryoSPARCC14.1FSC 0.143 CUT-OFFcryoSPARC2.51012085RANDOM ASSIGNMENTcryoSPARC2.5CryosparcMAXIMUM LIKELIHOODcryoSPARC2.55EPIFLEXIBLE FITREAL57