Cryo-EM structure of the CMG Fork Protection Complex at a replication fork - Conformation 1

PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork.

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

Polyoxyethylene (20) sorbitan monolaurate

Conformation 1 of CMG-Csm3-Tof1-Mrc1-Ctf4 with a fork DNA

Saccharomyces cerevisiae (strain ATCC 204508 / S288c)

In vitro reconstitution from individual components: CMG, Csm3-Tof1, Mrc1, Ctf4 and a DNA fork

Three microlitres of sample was applied on a grid and incubated for 15-30 s at 4 degC before manually blotting with filter paper for 10 s and plunge-freezing in liquid ethane.

DNA replication complex GINS protein PSF1

DNA replication complex GINS protein PSF2

DNA replication complex GINS protein PSF3

DNA replication complex GINS protein SLD5

Cy3-label at 5'-end. Five phosphorothioate backbone linkages between residues at the very 3'-end. The dT 16 residues from the 5'-end was biotinylated.

Chromosome segregation in meiosis protein 3

Cell division control protein 47,Minichromosome maintenance protein 7

Chromosome replication protein CHL15,Chromosome transmission fidelity protein 4,Protein POB1

MSDNRRRRREEDDSDSENELPPSSPQQHFRGGMNPVSSPIGSPDMINPEGDDNEVDDVPDIDEVEEQMNEVDLMDDNMYE
DYAADHNRDRYDPDQVDDREQQELSLSERRRIDAQLNERDRLLRNVAYIDDEDEEQEGAAQLDEMGLPVQRRRRRRQYED
LENSDDDLLSDMDIDPLREELTLESLSNVKANSYSEWITQPNVSRTIARELKSFLLEYTDETGRSVYGARIRTLGEMNSE
SLEVNYRHLAESKAILALFLAKCPEEMLKIFDLVAMEATELHYPDYARIHSEIHVRISDFPTIYSLRELRESNLSSLVRV
TGVVTRRTGVFPQLKYVKFNCLKCGSILGPFFQDSNEEIRISFCTNCKSKGPFRVNGEKTVYRNYQRVTLQEAPGTVPPG
RLPRHREVILLADLVDVSKPGEEVEVTGIYKNNYDGNLNAKNGFPVFATIIEANSIKRREGNTANEGEEGLDVFSWTEEE
EREFRKISRDRGIIDKIISSMAPSIYGHRDIKTAVACSLFGGVPKNVNGKHSIRGDINVLLLGDPGTAKSQILKYVEKTA
HRAVFATGQGASAVGLTASVRKDPITKEWTLEGGALVLADKGVCLIDEFDKMNDQDRTSIHEAMEQQSISISKAGIVTTL
QARCSIIAAANPNGGRYNSTLPLAQNVSLTEPILSRFDILCVVRDLVDEEADERLATFVVDSHVRSHPENDEDREGEELK
NNGESAIEQGEDEINEQLNARQRRLQRQRKKEEEISPIPQELLMKYIHYARTKIYPKLHQMDMDKVSRVYADLRRESIST
GSFPITVRHLESILRIAESFAKMRLSEFVSSYDLDRAIKVVVDSFVDAQKVSVRRQLRRSFAIYTLGH

MEGSTGFDGDATTFFAPDAVFGDRVRRFQEFLDTFTSYRDSVRSIQVYNSNNAANYNDDQDDADERDLLGDDDGDDLEKE
KKAASSTSLNILPHRIIISLDDLREFDRSFWSGILVEPAYFIPPAEKALTDLADSMDDVPHPNASAVSSRHPWKLSFKGS
FGAHALSPRTLTAQHLNKLVSVEGIVTKTSLVRPKLIRSVHYAAKTGRFHYRDYTDATTTLTTRIPTPAIYPTEDTEGNK
LTTEYGYSTFIDHQRITVQEMPEMAPAGQLPRSIDVILDDDLVDKTKPGDRVNVVGVFKSLGAGGMNQSNSNTLIGFKTL
ILGNTVYPLHARSTGVAARQMLTDFDIRNINKLSKKKDIFDILSQSLAPSIYGHDHIKKAILLMLMGGVEKNLENGSHLR
GDINILMVGDPSTAKSQLLRFVLNTASLAIATTGRGSSGVGLTAAVTTDRETGERRLEAGAMVLADRGVVCIDEFDKMTD
VDRVAIHEVMEQQTVTIAKAGIHTTLNARCSVIAAANPVFGQYDVNRDPHQNIALPDSLLSRFDLLFVVTDDINEIRDRS
ISEHVLRTHRYLPPGYLEGEPVRERLNLSLAVGEDADINPEEHSNSGAGVENEGEDDEDHVFEKFNPLLQAGAKLAKNKG
NYNGTEIPKLVTIPFLRKYVQYAKERVIPQLTQEAINVIVKNYTDLRNDDNTKKSPITARTLETLIRLATAHAKVRLSKT
VNKVDAKVAANLLRFALLGEDIGNDIDEEESEYEEALSKRSPQKSPKKRQRVRQPASNSGSPIKSTPRRSTASSVNATPS
SARRILRFQDDEQNAGEDDNDIMSPLPADEEAELQRRLQLGLRVSPRRREHLHAPEEGSSGPLTEVGTPRLPNVSSAGQD
DEQQQSVISFDNVEPGTISTGRLSLISGIIARLMQTEIFEEESYPVASLFERINEELPEEEKFSAQEYLAGLKIMSDRNN
LMVADDKVWRV

MSQQSSSPTKEDNNSSSPVVPNPDSVPPQLSSPALFYSSSSSQGDIYGRNNSQNLSQGEGNIRAAIGSSPLNFPSSSQRQ
NSDVFQSQGRQGRIRSSASASGRSRYHSDLRSDRALPTSSSSLGRNGQNRVHMRRNDIHTSDLSSPRRIVDFDTRSGVNT
LDTSSSSAPPSEASEPLRIIWGTNVSIQECTTNFRNFLMSFKYKFRKILDEREEFINNTTDEELYYIKQLNEMRELGTSN
LNLDARNLLAYKQTEDLYHQLLNYPQEVISIMDQTIKDCMVSLIVDNNLDYDLDEIETKFYKVRPYNVGSCKGMRELNPN
DIDKLINLKGLVLRSTPVIPDMKVAFFKCNVCDHTMAVEIDRGVIQEPARCERIDCNEPNSMSLIHNRCSFADKQVIKLQ
ETPDFVPDGQTPHSISLCVYDELVDSCRAGDRIEVTGTFRSIPIRANSRQRVLKSLYKTYVDVVHVKKVSDKRLDVDTST
IEQELMQNKVDHNEVEEVRQITDQDLAKIREVAAREDLYSLLARSIAPSIYELEDVKKGILLQLFGGTNKTFTKGGRYRG
DINILLCGDPSTSKSQILQYVHKITPRGVYTSGKGSSAVGLTAYITRDVDTKQLVLESGALVLSDGGVCCIDEFDKMSDS
TRSVLHEVMEQQTISIAKAGIITTLNARSSILASANPIGSRYNPNLPVTENIDLPPPLLSRFDLVYLVLDKVDEKNDREL
AKHLTNLYLEDKPEHISQDDVLPVEFLTMYISYAKEHIHPIITEAAKTELVRAYVGMRKMGDDSRSDEKRITATTRQLES
MIRLAEAHAKMKLKNVVELEDVQEAVRLIRSAIKDYATDPKTGKIDMNLVQTGKSVIQRKLQEDLSREIMNVLKDQASDS
MSFNELIKQINEHSQDRVESSDIQEALSRLQQEDKVIVLGEGVRRSVRLNNRV

MSFDRPEIYSAPVLQGESPNDDDNTEIIKSFKNFILEFRLDSQFIYRDQLRNNILVKNYSLTVNMEHLIGYNEDIYKKLS
DEPSDIIPLFETAITQVAKRISILSRAQSANNNDKDPENTSMDTDSLLLNSLPTFQLILNSNANQIPLRDLDSEHVSKIV
RLSGIIISTSVLSSRATYLSIMCRNCRHTTSITINNFNSITGNTVSLPRSCLSTIESESSMANESNIGDESTKKNCGPDP
YIIIHESSKFIDQQFLKLQEIPELVPVGEMPRNLTMTCDRYLTNKVIPGTRVTIVGIYSIYNSKNGAGSGRSGGGNGGSG
VAIRTPYIKILGIQSDVETSSIWNSVTMFTEEEEEEFLQLSRNPKLYEILTNSIAPSIFGNEDIKKAIVCLLMGGSKKIL
PDGMRLRGDINVLLLGDPGTAKSQLLKFVEKVSPIAVYTSGKGSSAAGLTASVQRDPMTREFYLEGGAMVLADGGVVCID
EFDKMRDEDRVAIHEAMEQQTISIAKAGITTVLNSRTSVLAAANPIYGRYDDLKSPGDNIDFQTTILSRFDMIFIVKDDH
NEERDISIANHVINIHTGNANAMQNQQEENGSEISIEKMKRYITYCRLKCAPRLSPQAAEKLSSNFVTIRKQLLINELES
TERSSIPITIRQLEAIIRITESLAKLELSPIAQERHVDEAIRLFQASTMDAASQDPIGGLNQASGTSLSEIRRFEQELKR
RLPIGWSTSYQTLRREFVDTHRFSQLALDKALYALEKHETIQLRHQGQNIYRSGV

MSSPFPADTPSSNRPSNSSPPPSSIGAGFGSSSGLDSQIGSRLHFPSSSQPHVSNSQTGPFVNDSTQFSSQRLQTDGSAT
NDMEGNEPARSFKSRALNHVKKVDDVTGEKVREAFEQFLEDFSVQSTDTGEVEKVYRAQIEFMKIYDLNTIYIDYQHLSM
RENGALAMAISEQYYRFLPFLQKGLRRVVRKYAPELLNTSDSLKRSEGDEGQADEDEQQDDDMNGSSLPRDSGSSAAPGN
GTSAMATRSITTSTSPEQTERVFQISFFNLPTVHRIRDIRSEKIGSLLSISGTVTRTSEVRPELYKASFTCDMCRAIVDN
VEQSFKYTEPTFCPNPSCENRAFWTLNVTRSRFLDWQKVRIQENANEIPTGSMPRTLDVILRGDSVERAKPGDRCKFTGV
EIVVPDVTQLGLPGVKPSSTLDTRGISKTTEGLNSGVTGLRSLGVRDLTYKISFLACHVISIGSNIGASSPDANSNNRET
ELQMAANLQANNVYQDNERDQEVFLNSLSSDEINELKEMVKDEHIYDKLVRSIAPAVFGHEAVKKGILLQMLGGVHKSTV
EGIKLRGDINICVVGDPSTSKSQFLKYVVGFAPRSVYTSGKASSAAGLTAAVVRDEEGGDYTIEAGALMLADNGICCIDE
FDKMDISDQVAIHEAMEQQTISIAKAGIHATLNARTSILAAANPVGGRYNRKLSLRGNLNMTAPIMSRFDLFFVILDDCN
EKIDTELASHIVDLHMKRDEAIEPPFSAEQLRRYIKYARTFKPILTKEARSYLVEKYKELRKDDAQGFSRSSYRITVRQL
ESMIRLSEAIARANCVDEITPSFIAEAYDLLRQSIIRVDVDDVEMDEEFDNIESQSHAASGNNDDNDDGTGSGVITSEPP
ADIEEGQSEATARPGTSEKKKTTVTYDKYVSMMNMIVRKIAEVDREGAEELTAVDIVDWYLLQKENDLGSLAEYWEERRL
AFKVIKRLVKDRILMEIHGTRHNLRDLENEENENNKTVYVIHPNCEVLDQLEPQDSS

MSAALPSIQLPVDYNNLFNEITDFLVTFKQDTLSSDATRNENEDENLDAENIEQHLLEKGPKYMAMLQKVANRELNSVII
DLDDILQYQNEKFLQGTQADDLVSAIQQNANHFTELFCRAIDNNMPLPTKEIDYKDDVLDVILNQRRLRNERMLSDRTNE
IRSENLMDTTMDPPSSMNDALREVVEDETELFPPNLTRRYFLYFKPLSQNCARRYRKKAISSKPLSVRQIKGDFLGQLIT
VRGIITRVSDVKPAVEVIAYTCDQCGYEVFQEVNSRTFTPLSECTSEECSQNQTKGQLFMSTRASKFSAFQECKIQELSQ
QVPVGHIPRSLNIHVNGTLVRSLSPGDIVDVTGIFLPAPYTGFKALKAGLLTETYLEAQFVRQHKKKFASFSLTSDVEER
VMELITSGDVYNRLAKSIAPEIYGNLDVKKALLLLLVGGVDKRVGDGMKIRGDINVCLMGDPGVAKSQLLKAICKISPRG
VYTTGKGSSGVGLTAAVMKDPVTDEMILEGGALVLADNGICCIDEFDKMDESDRTAIHEVMEQQTISISKAGINTTLNAR
TSILAAANPLYGRYNPRLSPLDNINLPAALLSRFDILFLMLDIPSRDDDEKLAEHVTYVHMHNKQPDLDFTPVEPSKMRE
YIAYAKTKRPVMSEAVNDYVVQAYIRLRQDSKREMDSKFSFGQATPRTLLGIIRLSQALAKLRLADMVDIDDVEEALRLV
RVSKESLYQETNKSKEDESPTTKIFTIIKKMLQETGKNTLSYENIVKTVRLRGFTMLQLSNCIQEYSYLNVWHLINEGNT
LKFVDDGTMDTDQEDSLVSTPKLAPQTTASANVSAQDSDIDLQDA

MYGDLGNKLVLEAKRTKQLYARSNQDVNLPMYHEDIIRNILKEVSNLRKNTEYLKEQQQLGMLDDKVAKCQYFVTLLCME
RNKRCLLAYQRLRTDILDSMAWNNNGLDLMSSITFSQQDTNNLSHQEQEYLKEYCDLITDLKSGDLVDIDLSGSLVPPSD
VFIDVRVLKDAGEIQTEYGVFNLIKDSQFFVRQSDVERLIQQGYLQKI

MSLPAHLQQTFSPEEIQFIVENEPIKIFPRITTRQKIRGDDRGTGNHTRWQLITTDDKALNNMVAMRSTEVVLWIALLLK
QQSKCSIVAPQWLTTKELDRKIQYEKTHPDRFSELPWNWLVLARILFNKAKDDFHDPIHELRGKIQDLREIRQIKVLKGL
KYLNESHLQLDNLSLLEINELRPFITEIMDKLREIHTASLTAGTENDEEEFNI

MGSSHHHHHHSSGLVPRGSHMASMGYYDIDDVLADGTEFPCKFQYDIPGLGYLENNPGRPITKNTKLSLPLWLARILAIV
GGDEALVDEEPVPFVELLPPDMFSTKVMNAIKTDPVALDLHSINSHFFSLAIKWIMLFSEKELANVVSELLLQRAQELNH
HASSLSIDLNADSTGKNSANTNIATSTFLLKLEEMEKEIYKKSHESYKDTKRWMFKK

MDINIDDILAELDKETTAVDSTKITQGSSSTTHRDANTIVGSSLDLNDKTQIYVSPQQDFSDLMKSWKNERCSPELLPYP
HQLMKRLLNRISMQSQLIENISMGFLDMQNASNANPPMPNESKLPLLCMETELERLKFVIRSYIRCRLSKIDKFSLYLRQ
LNEDENSLISLTDLLSKDEIKYHDTHSLIWLKLVNDSILKYMPEELQAINDTEGSVNMIDEPDWNKFVFIHVNGPPDGKW
NEDPLLQENEFGKPCYTVTIPDLKEEVELTIGSIYVMRYEVIRDLLRDDKVALI

MYYGISQFSEAYNKILRNSSSHSSCQLVIFVSCLNIDALCATKMLSLLFKKQLVQSQIVPIFGYSELRRHYSQLDDNINS
LLLVGFGGVIDLEAFLEIDPQEYVIDTDEKSGEQSFRRDIYVLDAHRPWNLDNIFGSQIIQCFDDGTVDDTLGEQKEAYY
KLLELDEESGDDELSGDENDNNGGDDEATDADEVTDEDEEDEDETISNKRGNSSIGPNDLSKRKQRKKQIHEYEGVLEEY
YSQGTTVVNSISAQIYSLLSAIGETNLSNLWLNILGTTSLDIAYAQVYNRLYPLLQDEVKRLTPSSRNSVKTPDTLTLNI
QPDYYLFLLRHSSLYDSFYYSNYVNAKLSLWNENGKKRLHKMFARMGIPLSTAQETWLYMDHSIKRELGIIFDKNLDRYG
LQDIIRDGFVRTLGYRGSISASEFVEALTALLEVGNSTDKDSVKINNDNNDDTDGEEEEDNSAQKLTNLRKRWVSNFWLS
WDALDDRKVELLNRGIQLAQDLQRAIFNTGVAILEKKLIKHLRIYRLCVLQDGPDLDLYRNPLTLLRLGNWLIECCAESE
DKQLLPMVLASIDENTDTYLVAGLTPRYPRGLDTIHTKKPILNNFSMAFQQITAETDAKVRIDNFESSIIEIRREDLSPF
LEKLTLSGLL

MVSVIDKLVFDFGGKTLVSLAPDNNTLCVANKNGLTKILKTNNPEEEPETLDSSKLVSSIKCYSNSHFLMTTMQGDALRY
NIDSSQEELLARFALPLRDCCVIHSGKMAVFGGDDLELILLELDDETHKKHAIKIDEQVSQISYNSQMNILAVSMINGKV
QIFSLTSTIPNKVHELNDYIVANSYDDTHRDKILSNMMDDIDKDNDNDLSETADPDENNVADPEFCAANRICTRVAWHPK
GLHFALPCADDTVKIFSIKGYSLQKTLSTNLSSTKAHFIDLQFDPLRGTYIAAVDLNNKLTVWNWETSEIHYTREFKRKI
TNIAWKIQADSKTLDLVLGTWSGSIAIVQNLAESVVSNIPDQSVAESSTKHGLFVDSESDLENLEGNDDINKSDKLFSDI
TQEANAEDVFTQTHDGPSGLSEKRKYNFEDEEDFIDDDDGAGYISGKKPHNEHSYSRVHKTHSFPISLANTGKFRYMPFS
PAGTPFGFTDRRYLTMNEVGYVSTVKNSEQYSITVSFFDVGRFREYHFEDLFGYDLCFLNEKGTLFGQSKTGQIQYRPHD
SIHSNWTKIIPLQAGERITSVAATPVRVIVGTSLGYFRSFNQFGVPFAVEKTSPIVALTAQNYRVFSVHYSQFHGLSYSL
SELGTSSKRYYKRECPLPMSLPNINSDMKKDANLDYYNFNPMGIKSLFFSSYGDPCIFGSDNTLLLLSKWRSPEESKWLP
ILDSNMEIWKMSGGKETTDIHVWPLALAYDTLNCILVKGKHIWPEFPLPLPSEMEIRMPVFVKSKLLEENKAILNKKNEI
GADTEAEEGEEDKEIQIPVSMAAEEEYLRSKVLSELLTDTLENDGEMYGNENEVLAALNGAYDKALLRLFASACSDQNVE
KALSLAHELKQDRALTAAVKISERAELPSLVKKINNIREARYEQQLK

(DT)(DA)(DG)(DA)(DG)(DT)(DA)(DG)(DG)(DA)(DA)(DG)(DT)(DG)(DA)(DT)(DG)(DG)(DT)(DA)
(DA)(DG)(DT)(DG)(DA)(DT)(DT)(DA)(DG)(DA)(DG)(DA)(DA)(DT)(DT)(DG)(DG)(DA)(DG)(DA)
(DG)(DT)(DG)(DT)(DG)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)
(DT)(DT)(DT)(DT)(DT)

TAGAGTAGGAAGTGATGGTAAGTGATTAGAGAATTGGAGAGTGTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT
TTTTT

(DG)(DG)(DC)(DA)(DG)(DG)(DC)(DA)(DG)(DG)(DC)(DA)(DG)(DG)(DC)(DA)(DC)(DA)(DC)(DA)
(DC)(DT)(DC)(DT)(DC)(DC)(DA)(DA)(DT)(DT)(DC)(DT)(DC)(DT)(DA)(DA)(DT)(DC)(DA)(DC)
(DT)(DT)(DA)(DC)(DC)(DA)(DT)(DC)(DA)(DC)(DT)(DT)(DC)(DC)(DT)(DA)(DC)(DT)(DC)(DT)
(DA)

MSADLQQGTTNAADFSLTVLRARIALLATAIGGPDYTSQIDPPPYKLGDDCLACLKDLKRWFKLVDDQQKRWDVAMAVAE
YRILTDDLLPILIDWENKCSLAAKLAKNNPDHEEFRNKAYYDKIALNCLQLLVLMTWPLIVTEQSSSNQITLYGELKKHQ
LVYKKTILSMESGKVLRAAIRLALDVIKIDRLSRTPRDNMVLKLVLNFFRNVIAIEPGEFTINTKKSMPKKGITSIDTLP
PNVSMDDISLNTVISSFHKNKVFGFLLTLTSSLSKEFDQDFINIPLLEIMFYFTKDVNQELLFPRQFETGTHSKVVNKNE
SSSANNIVTSAGFELSKLLQKEHQMRKNVIKHTSARHSRFGGLLSIQTPDKTRLTVSGSQALVDEKIALQKLDDSKKWNK
RIIKKHQSVAAEGLPNSLLNSQTGKAIFFTESNGKHFKEFINNFIDSGFNILLHSVTNYFTTEQDRMVTLEQVEYLLFFA
WFVKYQLLRSKIDNSADIKQVSEALKEVTFILVSSLLRSAYDLKNWTVTHAGMIAFNELLNLVSRTKAAQEEDSTDIEFI
VSRLFSDERIQLLSNLPKIGSKYSLQFMKSCIELTHSVLKVLEQYSDDKTLVIEGKSRRQKKFNISEGDITKLIEEENVD
RDEALDILTSSLRSIEVNFQKVQANYMTEPVIETYINFLERFRELEDDSIKKVFSFFHRVFVQAKEQALLFRFDLIILLR
EMLSPDGLDRMSRSRKYVSQFSDYFLARLKKRLKKSPAWFVGLLFPPLHNSEVGFYQRYGEYNVLNNESMYAAPASQFKP
IPDEEALPPSILLDMKYGVLVSTLLDDGKTELLDQLLKHITHTLDIFKSWLTVNVNAGKETVNPPNEYFTLTGVLNNDPI
FKDKDYRALLLLIGYSIPRKINEPCFLPGTVEVSDLTVSCELVKKYLSTPFETPNGLPSSSYLLRVRSEKDSFSHNEQDG
WEGDDDYDYNDPYIVPDDQILSKSDAAYFKDLDNNASDKLKGTKFSKGIARSKKKDKRKRRKGEAKTNLPMFGDQDDERP
QTVRERHGVFSKEFISDSEDDEDLMNPIFFENETYMRWLLDKNNGQLTEDRYIQFAKFAAERMNNGGVVTGDYTSLFGGS
IPSIESIRATESSSFAPDKSLISLASHVASEMSIFDVNNNNNNQLSDDDVNSESRNSLGSSQPSNSQNMFQSEVYSRKES
TKRSLEASAADESDEDEEAIRLFGKKSRVVLSQGDSDD

MDQDFDSLLLGFNDSDSVQKDPTVPNGLDGSVVDPTIADPTAITARKRRPQVKLTAEKLLSDKGLPYVLKNAHKRIRISS
KKNSYDNLSNIIQFYQLWAHELFPKAKFKDFMKICQTVGKTDPVLREYRVSLFRDEMGMSFDVGTRETGQDLERQSPMVE
EHVTSAEERPIVADSFAQDKRNVNNVDYDNDEDDDIYHLSYRNRRGRVLDERGNNETVLNNVVPPKEDLDALLKTFRVQG
PVGLEENEKKLLLGWLDAHRKMEKGSMTEEDVQLIQSLEEWEMNDIEGQHTHYDLLPGGDEFGVDQDELDAMKEMGF

Medical Research Council (MRC, United Kingdom)
Cryo-EM structure of a 1.4 MDa eukaryotic replisome complex at a replication fork - Conformation 1
cross-linking mass spectrometry complements our structural analysis