A/duck/Fujian/01/02. A/New_Territory/60/68. SLS. GMG. DLG. DLG. DLG. 5 5 0. 5 60 .... TV HYPK KTY EKVE LKHGTFGPVH RNQVKI R DVN GHADLSAK ADVI.
Molecular Cell, Volume 70
Supplemental Information
A Mechanism for the Activation of the Influenza Virus Transcriptase Itziar Serna Martin, Narin Hengrung, Max Renner, Jane Sharps, Mónica MartínezAlonso, Simonas Masiulis, Jonathan M. Grimes, and Ervin Fodor
A
B
C
Figure S1. Detailed view of pS5-CTD binding sites on FluPolC. (A) Detailed view of the pS5-CTD peptide inside the omit electron density map (Sigma-A weighted Fo-Fc, 2.5σ, green mesh) for pS5-CTD binding site 1. (B) Same as (A) for pS5-CTD binding site 2. (C) Ribbon representation of the pS5-CTD binding site 2 in the P3 C-ter domain involving a groove formed by P3 β6 and the loop between P3 β11 and β12. The backbone of the 10residue poly-alanine chain built is shown in yellow. Colours of FluPolC subunits are as in Figure 1. Related to Figure 1.
α1
α2
α3
β1
C/Johannesburg/1/66 1
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
10
20
30
40
50
α4
α5
β2
80
β3
90
α6
100
110
η1 .
β5
α7
TT 130
140
150
π1
α8
α9
α10
α11
TT 160
170
180
190
200
210
220
230
240
ATAADFLRKENMFNEIFLPDNEETIIE..MKKGKTFLELRDESVPLPFQTYEQMKDYCEKFKGNPRELASKVSQMQSNIKLPIKHYEQNK TELQAELSLKNLWQVLIGEEDVEKGID..FKLGQTISRLRDISVPAGFSNFEGMRSYIDNIDPKG.AIERNLARMSPLVSATPKKLKWED FVLRQELATAGLWDSFRQSEKGEETLEEEFSYPPTFQRLANQSLPPSFKDYHQFKAYVSSFKANG.NIEAKLGAMSEKVNAQIESFDPRT FTIRQEMASRGLWDSFRQSERGEETIEERFEITGTMRRLADQSLPPNFSSLENFRAYVDGFEPNG.CIEGKLSQMSKEVNARIEPFLKTT FTIRQEMANRGLWDSFRQSERGEETIEERFEITGTMRRLADQSLPPNFSCLENFRAYVDGFEPNG.YIEGKLSQMSKEVNAKIEPFLKTT
β6 C/Johannesburg/1/66
α12
α13 .......
T.T 250
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
β4
120
TAVSLQNELCKSYDLEPLPFLCDIFDREEKQFVEIGITRKADDSYFQSKFGKLG.NSCKIFVFSYDGR.LDKNCEGPM.EEQKLRIFSFL IAWMVQRSLAQEHGIETPKYLADLFDYKTKRFIEVGITKGLADDYFWKKKEKLG.NSMELMIFSYNQD.YSLSNESSLDEEGKGRVLSRL IAWTIVNSICNMTENSKPRFLPDLYDYKTNKFIEIGVTRRKVEDYYYEKASKLKGENVYIHIFSFDGEEMATDDEYILDEESRARIKTRL MAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKSEKTHIHIFPFTGEEMATKADYTLDEESRARIKTRL MAWTVVNSICNTTGAEKPKFLPDLYDYKENRFIEIGVTRREVHIYYLEKANKIKSENTHIHIFSFTGEEMATKADYTLDEESRARIKTRL
C/Johannesburg/1/66 C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
70
MSKTFAEIAEAFLEPEAVRIAKEAVEEYGDH....ERKIIQIGIHFQVCCMFCDEY.LSTNG...............SDRFVLIEGRKRG MDTFI....TRNFQTTIIQKAKNTMAEFSEDPELQPAMLFNICVHLEVCYVISDMNFLDEEGKSYTALEGQGKEQNLRPQYEVIEGMPRT MENFV....RTNFNPMILERAEKTMKEYGENPQNEGNKFAAISTHMEVCFMYSDFHFIDLEGNTIVK.ENDDDNAMLKHRFEIIEGQERN MEDFV....RQCFNPMIVELAEKAMKEYGEDPKIETNKFAAICTHLEVCFMYSDFHFIDERGESIIV.ESGDPNALLKHRFEIIEGRDRT MEDFV....RQCFNPMIVELAEKAMKEYGEDLKIETNKFAAICTHLEVCFMYSDFHFINEQGESIVV.ELDDPNALLKHRFEIIEGRDRT
C/Johannesburg/1/66 C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
60
260
270
280
η2 TT 290
300
. 310
FRQI.......RLPKGPMAPYTHKFLME.EAWMFTKISDPERSRAGEILI.......DFFKKG...NLSAIRPKDKPLQGKYP.IHYKNL LRPIGPHIYNHELPEV...PY.NAFLLMSDELGL...ANMTEGKSKKPKTLAKECLEKYSTLRDQTDPILIMKSEKA.NENFLWKLWRDC IREL.......ELPEGKFCTQRSKFLLM.DAMKLS.VLNPAHEGEGIPMKDAKACLDTFWGWK...KATIIKKHEKGVNTNYL.MIWEQL PRPL.......RLPDGPPCSQRSKFLLM.DALKLS.IEDPSHEGEGIPLYDAIKCMKTFFGWK...EPNIIKPHEKGINPNYL.LAWKQV PRPI.......RLPDGPPCFQRSKFLLM.DALKLS.IEDPSHEGEGIPLYDAIKCMRTFFGWK...EPYIVKPHEKGINPNYL.LSWKQV
α14
α15
β7
α16
α17
C/Johannesburg/1/66 320
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
330
340
350
360
370
380
390
400
WNQIKAAIAD....RTMVINENDHSEFLGGIGRASKKIPEISLTQDVITTEGLKQSENKLPEPRSFPRWFNAEWMWAIKDSDLTGWVPMA VNTI...SNE....MSNELQKTNYAKWATGDGLTYQKI....MKEVAIDDETMCQEEPKIPNKCRVAAWVQTEMNLLSTLTS.KRALDLP LESIKEMEG.....KFLNLKKTNHLKWGLGEGQAPEKM....DFEDCKEVPDLFQYKSEPPEKRKLASWIQSEFNKASELTN.SNWIEFD LAELQDIENEEKIPKKKNMKKTSQLKWALGENMAPEKV....DFEDCKDVSDLKQYDSDEPEPRSLASWIQSEFNKACELTD.SSWIELD LAELQDIENEEKIPRTKNMKKTSQLKWALGENMAPEKV....DFDNCRDVSDLKQYDSDEPELRSLSSWIQNEFNKACELTD.STWIELD
α18
α19
β8
β9
C/Johannesburg/1/66 410
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
420
430
440
450
460
470
480
490
EYPPADNELEDYAEHLNKTMEGVLQGTNCAREMGKCILTVGALMTECRLFPGKIKVVPIYARSKERKSMQEGLPVPSEMDCLFGICVKSK EIGPDVAPVEHVGSERRKYFVNEINYCKASTVMMKYVLFHTSLLNESNASMGKYKVIPITNRVVNEKG........ESFDMLYGLAVKGQ ELGNDVAPIEHIASRRRNFFTAEVSQCRASEYIMKAVYINTALLNSSCTAMEEYQVIPIITKCRDTSG........QRRTNLYGFIIKGR EIGEDVAPIEHIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCRTKEG........RRKTNLYGFIIKGR EIGEDVAPIEYIASMRRNYFTAEVSHCRATEYIMKGVYINTALLNASCAAMDDFQLIPMISKCRTKEG........RRKTNLYGFIIKGR
β10
β11
β12
α20
α21
C/Johannesburg/1/66 500
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
510
530
540
550
560
570
580
SHLNKDDGMYTIITFEFSIREPNLEKH..QKYTVFEAGHTTVRMKKGESVIGREVPLYLYCRTTALSKIKNDWLSKARRCFITTMDTVET SHLRGDTDVVTVVTFEFSSTDPRVDSGKWPKYTVFRIGSLFVS........GREKSVYLYCRVNGTNKIQMKWGMEARRCLLQSMQQMEA SHLRNDTDVVNFISLEFSLTDPRNEIHKWEKYCVLEIGDMEIRTSIS....TIMKPVYLYVRTNGTSKIKMKWGMEMRRCLLQSLQQVES SHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRTAIG....QVSRPMFLYVRTNGTSKIKMKWGMEMRRCLLQSLQQIES SHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCVLEIGDMLLRSAIG....QMSRPMFLYVRTNGTSKIKMKWGMEMRRCLLQSLQQIES
α22 .....
C/Johannesburg/1/66 590
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
520
β13 .
600
β14
α23
α24
β15
TT 610
620
630
640
650
660
ICLRESAKAEENLVEKTL.....NEKQMW.IGKKNGELIAQPLREALRVQLVQQFYFCIYNDSQLEGFCNEQKKILMALEGDKKNKSSFG IVEQESSIQGYDMTKACFKGDRVNSPKTFSIGTQEGKLVKGSFGKALRVIFTKCLMHYVFGNAQLEGFSAESRRLLLLIQALKDRKGPWV MIEAESAVKEKDMTEPFFR....NRENDWPIGESPQGIEKGTIGKVCRVLLAKSVFNSIYASAQLEGFSAESRKLLLLIQAFRDNLDPGT MIEAESSVKEKDMTKEFFE....NKSETWPIGESPKGVEEGSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRRLLLIVQALRDNLEPGT MIEAESSVKEKDMTKEFFE....NKSETWPIGESPKGVEDGSIGKVCRTLLAKSVFNSLYASPQLEGFSAESRKLLLVVQALRDNLEPGT
α25
α26
C/Johannesburg/1/66 670
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
680
690
700
FNPEGLLEKIEECLINNPMCLFMAQRLNELVIEASKRGAKFFKTD...... FDLEGMYSGIEECISNNPWVIQSAYWFNEWLGFE.KEGSKVLESVDEIMDE FDLKGLYEAIEECIINDPWVLLNASWFNSFLKAV.Q..........LSM.. FDLGGLYEAIEECLINDPWVLLNASWFNSFLTHA.L............R.. FDLEGLYEAIEECLINDPWVLLNASWFNSFLTHA.L............R..
Figure S2. Amino acid sequence alignment of FluPol PA/P3 subunit. Amino acid sequence alignment for the PA/P3 subunit from influenza C/Johannesburg/1/66, B/Panama/45/90, A/bat/Guatemala/060/10, A/duck/Fujian/01/02 and A/New Territory/60/68. Highly conserved residues are shown in red, partially conserved residues in yellow. Amino acid numbering and secondary structure correspond to C/Johannesburg/1/66. Residues proximal to FluPolC pS5-CTD binding site 1 are highlighted by green arrows. The residues in FluPolA and FluPolB PA involved in pS5-CTD binding reported by Lukarska et al., 2017 are highlighted. Purple arrows indicate residues at site 1, which are conserved in influenza A and B viruses; blue arrows indicate residues at site 2, conserved only in influenza A viruses. Related to Figure 1 and Figure 2.
β1
η1
α1
η2
α2
C/Johannesburg/1/66
β2
β3
TT 1
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
10
TTT
20
30
40
50
60
70
80
90
MEINPYLMFLNNDVTSLISTTYPYTGPPPMSHGSSTKYTLETIKRTYDYSRTSVEKTSKVFNIPRRKFCNCLEDKDELVKPTGNVDISSL MNINPYFLFIDVPIQAAISTTFPYTGVPPYSHGTGTGHTIDTVIRTHEYSNKGKQYVSDVTGCTMVDPTNGPL..PEDNEPSAYAQLDCV MDVNPMLIFLKVPVQNAISTTFPYTGDPPYSHGTGTGYTMDTVIRTHDYSSRGIWKTNSETGAQQLNPIDGPL..PEDNEPSGYAQTDCV MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTETGAPQLNPIDGPL..PDNHEPSGYAQTDCV MDVNPTLLFLKVPAQNAISTTFPYTGDPPYSHGTGTGYTMDTVNRTHQYSEKGKWTTNTETGAPQLNPIDGPL..PEDNEPSGYAQTDCV
α3
α4
η3
β4
β5
α5
140
150
α6
C/Johannesburg/1/66 100
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
110
120
130
160
170
LGLAEMMEKRMGEGFFKHCVMEAETEILKMHFSRLTEGRQTYDWTSERNMPAATALQLTVDAIKETEGPF.KGTTMLEYCNKMIEMLDWK LEALDRMDEE.HPGLFQAASQNAMEALMVTTVDKLTQGRQTFDWTVCRNQPAATALNTTITSFRLNDLNGADKGGLVPFCQDIIDSLDKP LELIEGLDRS.HPGLFETACQETIDAIQQTRVDKLTQGRQTYDWTLNRNQPAATALANTIEVFRKNGYKLNESGRLIDFLKDVLLSFEND LEAMAFLEES.HPGIFENSCLETMEVVQQTRVDKLTQGRQTYDWTLNRNQPAATALANTIEVFRSNDLTANESGRLIDFLKDVMESMDKE LEAMAFLEES.HPGIFENSCLETMEVVQQTRVDRLTQGRQTYDWTLNRNQPAATALANTIEVFRSNGLTANESGRLIDFLKDVMESMDKE
β6
β7
α7
β8
β9
β10
α8
C/Johannesburg/1/66
TTT 180
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
190
200
210
220
230
240
250
260
EIKFKKVKTVVRREKDKRSGKEIKTKVPVMGIDSIKHDEFLIRALTINTMAKDGERGKLQRRAIATPGMIVRPFSKIVETVAQKICEKLK EMTFFSVKNIKKKLPAKNRKGFLIKRIPMKVKDRITRVEYIKRALSLNTMTKDAERGKLKRRAIATAGIQIRGFVLVVENLAKNICENLE SMEVTTHFQKKKRIRDNHSKKMITQRTIGKKRVKLTKKNYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVELLARNICERLE EMEITAHFQRKRRIRDNMTKKMVTQRTIGKKKQRLNKKSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLE EMEITTHFQRKRRVRDNMTKKMVTQRTIGKKKQRVNKRSYLIRALTLNTMTKDAERGKLKRRAIATPGMQIRGFVYFVETLARSICEKLE
α9
β11
α10
α11
α12
β12
β13
C/Johannesburg/1/66 270
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
280
290
β14
α13
TT 360
370
η4 .
α14
330
340
350
β15
390
α15
400
410
β16
420
430
TT 440
SNKRKTKEVIIKAEKMGKYK.NLMREEYKNLFEPLEKYIQ.KDVCFLPGGMLMGMFNMLSTVLGVSTLCYMDEELKAKGCFWTGLQSSDD TSKTKRLKAQIPCPDLFSIPLERYNEETRAKLKKLKPFFNEEGTASLSPGMMMGMFNMLSTVLGVAAL..GIKNIGNKEYLWDGLQSSDD ESKSMHLRTQISAENLSDINLRYFNEDTKKKIEKIRHLMV.EGTASLSPGMMMGMFNMLSTVLGVSVLNLGQREILKRTYWWDLLQSSDD ESKSMKLRTQIPAEMLANIDLKYFNESTRKKIEKIRPLLI.DGTASLSPGMMMGMFNMLSTVLGVSILNLGQKRYTKTTYWWDGLQSSDD ESKSMKLRTQIPAEMLASIDLKYFNESTRKKIEKIRPLLI.DGTASLSPGMMMGMFNMLSTVLGVSILNLGQKRYTKTTYWWDGLQSSDD
β17
α16
β18 TTT
450
460
470
β19 TT
480
β20
η5
η6
α17
TT
490
500
510
520
530
FVLFAVASNWSNIHWTIRRFNAVCKLIGINMSLEKSYGSLPELFEFTSMFFDGEFVSNLAMELPAFTTAGVNEGVDFTAAMSIIKTNMIN FALFVNAKDEETCMEGINDFYRTCKLLGINMSKKKSYCNETGMFEFTSMFYRDGFVSNFAMEIPSFGVAGVNESADMAIGMTIIKNNMIN FALIINGHFKEDIQQGVNHFYRTCKLVGINMSQKKSYINKTGTFEFTSFFYRYGFVANFSMELPSFGVAGNNESADMSIGTTVIKTNMIN FALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYINRTGTFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMIN FALIVNAPNHEGIQAGVDRFYRTCKLVGINMSKKKSYINRTGTFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIKNNMIN
α18
α19
C/Johannesburg/1/66
α20
α21
TT 540
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
320
TT
380
C/Johannesburg/1/66 C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
310
ESGLPVGGNEKKAKLKTTVTSLNARMNSDQFAVNITGDNSKWNECQQPEAYLALLAYITKDSSDLMKDLCSVAPVLFCNKFVKLGQGIRL QSGLPVGGNEKKAKLSNAVAKMLSNCPPGGISMTVTGDNTKWNECLNPRIFLAMTERITRDSPIWFRDFCSIAPVLFSNKIARLGKGFMI QSGLPVGGNEKKAKLANVIKKMMAKSTDEELSYTITGDNTKWNENQNPRIFLAMVLRITAGQPEWFRDLLAVAPIMFSNKVARLGRGYMF QSGLPVGGNEKKAKLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRMFLAMITYITRNQPDWFRNVLSIAPIMFSNKMARLGKGYMF QSGLPVGGNEKKAKLANVVRKMMTNSQDTELSFTITGDNTKWNENQNPRMFLAMITYITKNQPEWFRNVLSIAPIMFSNKMARLGKGYMF
C/Johannesburg/1/66 C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
300
550
560
570
580
590
600
610
620
NSLSPSTALMALRICLQEFRATYRVHPWDSRVKGGRMKIINEFIKTIENKDGLLIADGGKLMNNISTLHIPEEVLKFEKMDEQYRNRVFN NGMGPATAQTAIQLFIADYRYTYKCHRGDSKVEGKRMKIIKELWENTKGRDGLLVADGGPNIYNLRNLHIPEIVLKYNLMDPEYKGRLLH NDLGPATAQMAIQLFIKDYRYTYRCHRGDTNLETRRTKSIKRLWTETISKAGLLVADGGPNPYNLRNLHIPEVCLKWSLMDPDYRGRLCN NDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFELKKLWEQTRSKAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCN NDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTRRSFELKKLWEQTRSKAGLLVSDGGPNLYNIRNLHIPEVCLKWELMDEDYQGRLCN
α22 C/Johannesburg/1/66 630
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
640
650
660
670
680
690
700
710
PKNPFTNFDK.....TIDIFRAHGPIRVEENEAVVSTHSFRTRANRTLLNTDMRAMMAEEKRYQMVCDMFKSVFESADINPPIGAMSIGE PQNPFVGHLSIEGIKEADITPAHGPVKKMDYDAVSGTHSWRTKRNRSILNTDQRNMILEEQCYAKCCNLFEACFNSASYRKPVGQHSMLE PNNPFVHHMEVESTNLAVVMPAHGPAKSLEYDAVATTHSWTPKRNRSILNTNQRGILEDERIYQKCCQVFEKFFPSSTYRRPIGMASMLD PLNPFVSHKEIESANNAVVMPAHGPAKSMEYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFFPSSSYRRPVGISSMVE PLNPFVSHKEIESVNNAVVMPAHGPAKSMEYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQKCCNLFEKFFPSSSYRRPVGISSMVE
α23
α24
C/Johannesburg/1/66 720
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
730
740
750
AIEEKLLERAKMKRDIGAIEDSEYEEIKDIIRDAKKARLESR AMAHRLRMDARLDYESGRMSKDDFEKAMAHLGEIGYI..... AMLSRARIDARIDLESGRISSQDFSEITNTCKAIEALKR..Q AMVSRARIDARIDFESGRIKKEEFAEIMKICSTIEELRRQ.K AMVSRARIDARIDFESGRIKKEEFAEIMKICSTIEELRRQ.K
Figure S3. Amino acid sequence alignment of FluPol PB1 subunit. Amino acid sequence alignment for the PB1 subunit from influenza C/Johannesburg/1/66, B/Panama/45/90, A/bat/Guatemala/060/10, A/duck/Fujian/01/02 and A/New Territory/60/68. Highly conserved residues are shown in red, partially conserved residues in yellow. Amino acid numbering and secondary structure correspond to C/Johannesburg/1/66. Residues proximal to FluPolC pS5-CTD binding site 1 are highlighted by green arrows. Related to Figure 1 and Figure 2.
Figure S4. Cryo-EM analysis of FluPolC bound to vRNA promoter. (A) The structure of apo FluPolC in transcriptionally inactive conformation (PDB: 5D98) was fit into the cryo-EM density of FluPol. Two possible fits of the crystal structure are shown: in fit 1, the PA endonuclease domain is placed inside a small peripheral density, which leaves large areas of the density volume unoccupied (red arrow); in fit 2, the PB2 627 domain is placed inside the small density, leaving the endonuclease domain protruding outside the density. (B) Local resolution of the vRNA promoter bound FluPolC cryo-EM density map. Note that the third panel shows a distinct patch of low resolution. (C) Particle orientation distribution in the vRNA promoter bound FluPolC cryo-EM dataset showing two main populations with reduced coverage of other views. Orientation distribution plot generated in cryoSPARC (Punjani et al., 2017). Related to Figure 3.
α1 C/Johannesburg/1/66 C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
α2
α3
α4
α5 .
.. 1
10
20
30
40
50
60
70
80
MS..LLLTIAKEYKRLCQDAKAAQMMTVGTVSNYTTFKKWTTSRKEKNPSLRMRWAMSSKFPIIANKRML.EEAQIP.KEHNNVALWEDT MTLAK....IELLKQLLRDNEAKTVLKQTTVDQYNIIRKFNTSRIEKNPSLRMKWAMCSNFPLALTKGDMANR..IP.LEYKGIQLKTNA ME..R....IKELMEMVKNSRMREILTTTSVDHMAVIKKYTSGRQEKNPALRMKWMMAMKYPISASSRIR.EM..IPEKDEDGNTLWTNT ME..R....IKELRDLMSQSRTREILTKTTVDHIAIIKKYTSGRQEKNPALRMKWMMAMKYPITADKRIM.EM..IPERNEQGQTLWSKT ME..R....IKELRNLMSQSRTREILTKTTVDHMAIIKKYTSGRQEKNPSLRMKWMMAMKYPITADKRIT.EM..VPERNEQGQTLWSKM
α6
α7
η1
α8
β1
β2
α9
C/Johannesburg/1/66
TT 90
100
110
120
130
140
150
160
170
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
EDVSKRDHVLASASCINYWNFCGPCVNNSEVIKEVYKSRFGRLERRKEIMWKELRFTLVDRQRRRVDTQPVEQRLRTGEIKDLQMWTLFE EDIGT.KGQMCSIAAVTWWNTYGPIGD.TEGFEKVYESFFLRKMRLDNATWGRITFGPVERVRKRVLLNPLTKEMPPDEASNVIMEILFP KDAGS.NRVLVSPNAVTWWNRAGPVSD.VVHYPRVYKMYFDRLERLTHGTFGPVKFYNQVKVRKRVDINPGHKDLTSREAQEVIMEVVFP NDAGS.DRVMVSSLAVTWWNRNGPTTS.TVHYPKVYKTYFEKVERLKHGTFGPVHFRNQVKIRRRVDVNPGHADLSAKEAQDVIMEVVFP SDAGS.DRVMVSPLAVTWWNRNGPMTS.TVHYPKVYKTYFEKVERLKHGTFGPVHFRNQVKIRRRVDINPGHADLSAKEAQDVIMEVVFP
C/Johannesburg/1/66
TT
α10 180
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
α11 190
α12
200
α15
α16
α13 230
α14
β4
240
250
β5
β6
α17
α18
β7
TT 270
280
290
260
β8
β9
TT
300
310
320
330
340
350
RAVCRKVCLAANASIMNAKSKLVEYIKSTSMRIGETERKLEELILETDDVSPEVTLCKSALGGQLGKTLSFGPMLLKKISGSGVKVKDTV IVACRKIIRRSIVAS.NPLELAVEIANKTV..IDTE..PLKSCLTAIDGGDVACDIIRAALGLKIRQRQRFGRLELKRISGRGFKNDEEI IIASRNIVRRSIVAI.DPLASLLSMCHTTS..ISSE..PLVEILRSNPTDEQAVNICKAALGIRINNSFSFGGYNFKRVKGSSQRTEKAV IIAARNIVRRATVST.DPLASLLEMCHSTQ..IGGI..RMVDILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSVKKEEEV IIAARNIVRRAAVSA.DPLASLLEMCHSTQ..IGGT..RMVDILRQNPTEEQAVDICKAAMGLRISSSFSFGGFTFKRTSGSSIKREEEL 1 1 β10
β11
C/Johannesburg/1/66
β12
β13
TT 360
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
220
DEAPLASKFILDNYGLVKEMRSKFANKPLNKEVVAHMLEKQFNPESRFLPVFGAIRPERMELIHALGGETWIQEANTAGISNVDQRKNDI KEAGIPRESTWIHRELIKEKREKLKGTMITPIVLAYMLERELVARRRFLPVAGATSAEFIEMLHCLQGENWRQIYHPGGNKLTESRSQSM NEVGARTLSSDAQLTITKEKKEELKNCKISPIMVAYMLERELVRRTRFLPIAGATSSTYVEVLHLTQGTCWEQQYTPGGEAENDDLDQTL NEVGARILTSESQLTITKEKKEELQDCKIAPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSL NEVGARILTSESQLTITKEKKEELQDCKISPLMVAYMLERELVRKTRFLPVAGGTSSVYIEVLHLTQGTCWEQMYTPGGEVRNDDVDQSL
C/Johannesburg/1/66 C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
β3
210
370
α19
α20
β14
β15
TT
380
390
400
410
420
430
440
YIQGVRAVQFEYWSEQEEFYGEYKSATALFSRKERSLEWITIGGGINEDRKRLLAMCMIFCRDGDYFKDAPATITMADLSTKLGREIPYQ LIGNGTIQKIGIWDGEEEFHVRCGECRGILKKSKMRMEKLLINSAKKEDMKDLIILCMVFSQDTRMFQGVRGEINFLNRAGQ...LLSPM LTGNLQTLTMTIFEGYEEFNVSGKRASAVLKKGAQRLIQAIIGGRTLEDILNLMITLMVFSQEEKMLKAVRGDLNFVNRANQ...RLNPM LTGNLQTLKIRVHEGYEEFTMVGRRATAILRKATRRLIQLIVSGRDEQSIAEAIIVAMVFSQEDCMIKAVRGDLNFVNRANQ...RLNPM LTGNLQTLKIRVHDGYEEFTMVGKRATAILRKATRRLVQLIVSGRDEQSVAEAIIVAMVFSQEDCMIKAVRGDLNFVNRANQ...RLNPM
α21
α22
β16
β17
β18
β19
β20
C/Johannesburg/1/66 450
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
460
470
480
490
500
510
520
530
YVMMNWIQKSEDNLEALLYSRGIVETNPGKMGSSMGI..DGSKRAIKSLRAVTIQSGKIDMPESKEKIHLELSDNLEAFDSSGRIVATIL YQLQRYFLNRS...NDLFDQWGYEESPK.ASE.LHGINEL.MNASDYTLKGVVVTKNVIDDFSSTETEKVSITKNLSLIKRTGEVIMGAN YQLLRHFQKDS...STLLKNWGTEEIDP.IMG.IAGIMPDGTINKTQTLMGVRLSQGGVDEYSFNERIRVNIDKYLRVRNEKGELLISPE HQLLRHFQKDA...KVLFQNWGIEPIDN.VMG.MIGILPDMTPSTEMSLRGVRVSKMGVDEYSSTERVVVSIDRFLRVRDQRGNVLLSPE HQLLRHFQKDA...KVLFQNWGIEHIDN.VMG.MIGVLPDMTPSTEMSMRGIRVSKMGVDEYSSTERVVVSIDRFLRVRDQRGNVLLSPE
α23
α24
α25
α26
η2
η3
α27
C/Johannesburg/1/66 540
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
550
580
590
600
610
α28
η4
β21
620
β22
TT
TT
630
640
650
660
670
680
690
TT 700
TQV.TTGHYSLHELIKVLPFTYFAPKQGMF..EGRLFF.....SNDSFVEPGVNNNVFSWSKADSSKIYCHGIAIRVPLVV..GDEHMDT QMRDQ.EVMKTDQFIKLLPFCFSPPKLRSNGEPYQFLRLVLKGGGENFIEVRKGSPLFSYNPQTEVL.TICGRMMSLKGKIEDEERNRSM QMRDVEGTFDSIQIIKLLPFSAHPPSLGRT..QFSSFTLNIRGAPLR.LLIRGNSQVFNYNQMENVI.IVLGKSVGSPERSI.LTESSSI QMRDVVGTFDTVQIIKLLPFTAAPPEQSRM..QFSSLTVNVRGSGMR.ILVRGNSPVFNYNKATKRL.TVLGKDAGALTEDP.DEGTAGV QMRDVLGTFDTTQIIKLLPFAAAPPKQSRM..QFSSLTVNVRGSGMR.ILVRGNSPAFNYNKTTKRL.TILGKDAGTLIEDP.DEGTSGV
α29
β23
η5
C/Johannesburg/1/66
β24 TT
710
C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
570
DLPSDKKVTFQDVSFQHPDLAVLRDEKTAITKGYEALIKRLGTGDNDIPSLIAKKDYLSLYNLPEVKLMAPLIRPNRKGVYSRVARKLVS DVSELESQAQLMITYDTPKMWEMGTTKELVQNTYQWVLKNLVTLK.....AQFLLGKEDMFQWDAFEAFESIIPQKMAGQYSGFARAVLK EVSEAQGQEKLPINYNSSLMWEVNGPESILTNTYHWIIKNWELLK.....TQWMTDPTVLYNRIEFEPFQTLIPKGNRAIYSGFTRTLFQ EVSETQGTERLTITYSSSMMWEINGPESVLVNTYQWVIRNWETVK.....IQWSQDPTMLYNKMEFEPFQSLIPKAARGQYSGFVRTLFQ EVSETQGTEKLTITYSSSMMWEINGPESVLVNTYQWIIRNWETVK.....IQWSQNPTMLYNKMEFEPFQSLVPKAIRGQYSGFVRTLFQ
C/Johannesburg/1/66 C/Johannesburg/1/66 B/Panama/45/90 A/bat/Guatemala/060/10 A/duck/Fujian/01/02 A/New_Territory/60/68
560
720
730
β25
α30
TT 740
750
760
770
SLALLEGFSVCE..NDPRAPMVTRQDLIDVGFGQKVRLFVGQGSVRTFKRTASQRAASSDVNKNVKKIKMSN........ GNAVLAGFLVSGKYDPDLGDFKTIEELEKLKPGEKANILLYQGKPVKVVKRKRYSALSNDISQGIKRQRMTVESMGWALS ESAVLRGFLILGKANSKYGPVLTIGELDKLGRGEKANVLIGQGDTVLVMKRKRDSSILTDSQTALKRIRLEESK...... ESAVLRGFLILGKEDKRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAI.N...... ESAVLRGFLILGKEDRRYGPALSINELSNLAKGEKANVLIGQGDVVLVMKRKRDSSILTDSQTATKRIRMAI.N......
Figure S5. Amino acid sequence alignment of FluPol PB2 subunit. Amino acid sequence alignment for the PB2 subunit from influenza C/Johannesburg/1/66, B/Panama/45/90, A/bat/Guatemala/060/10, A/duck/Fujian/01/02 and A/New Territory/60/68. Highly conserved residues are shown in red, partially conserved residues in yellow. Amino acid numbering and secondary structure correspond to C/Johannesburg/1/66. Residues proximal to FluPolC pS5-CTD binding site 1 are highlighted by green arrows. Related to Figure 4 and Figure 5.
Relative(RNA(levels
1.5
**
1.0
0.5
0.0
** ** BG
'
5 '3 '
5 '3 '*p S 5
5 '3 '*p S 2
5 '3 '*U n P
5 '3 '*S c r
Figure S6. Effect of Pol II CTD peptides on FluPolA transcriptional activity. In vitro cap-dependent transcription initiation assay performed using purified FluPolA in the presence of pS5, pS2, un-phosphorylated (UnP) or scrambled (Scr) Pol II CTD peptides. Assay was carried out in the absence or presence of 5' and 3' vRNA promoter RNAs. The mean of three independent experiments is shown with error bars representing standard deviation. Asterisks indicate a significant difference from the reaction in absence of CTD peptide (**P < 0.01, based on a two-sample t-test). Related to Figure 6.