Structural insights into C3 convertase activity of the classical pathway of complement

Domain: chemistry
Published: 2025-12-18
Editorial outcome: major_revision

Abstract
ImmuneprotectionbythecomplementsystemdependsonC3cleavagebyC3convertasesthatiscriticaltoallthreeactivationpathways.StructuraldataonconvertaseformationintheclassicalpathwayandonC3-substratebindingtoconvertasesislacking.Wepresentthecryo-EMstructuresofthepro-convertase(C4b2),convertase(C4b2b),andconvertase-substratecomplex(C4b2b-C3)oftheclassicalpathway.ThedatashowthatC2andC4bformproconvertasesandconvertaseslikefactorBandC3bofthealternativepathway.SubstrateC3bindsC4boftheconvertasethroughtwointerfaces:onealsofoundintheSCIN-inhibitedC3bBbdimer,andanotherfacilitatedbyconformationalchangesinC3.BendingofC3andswingingoftheC2proteasebringtheC3-scissileloopintotheactivesite.Thesecond,charged,C4b-interactionsitefavorsC3-substratebinding,butuponcleavagerepelsproductC3b.Thus,achargeswitch-overmechanismeffectsthecatalyticturnoveroftheconvertasesproducingopsoninC3b.

Introduction
Thehumoralcomplementsysteminhumanshasimportantrolesintissuehomeostasisandimmuneprotectionagainstinfectiousagents1,2. A central and pivotal step in the complement response is labelling targetedcellsanddebrisforclearancebydepositionofC3bthroughC3 convertases that cleave C3 into anaphylatoxin C3a and opsonin C3b2,3.C3convertasesareirreversiblydissociating,bimolecularproteolyticcomplexeswithca.1–1.5min.half-lifetimes4,5.Theyaregeneratedonsurfacesbydanger-recognitioncomplexesofthe(so-called) classicalandlectinpathways,andthroughafeed-backamplificationloop of the alternative pathway1,6,7. The initiation complexes of the classical and lectin pathways first cleave complement C4 into anaphylatoxinC4aandC4b,whichbindscovalentlytothetargetedsurfacethroughitsthioestermoiety8,9.Next,pro-enzymeC2bindstoC4bformingaC3proconvertase,C4b2.Subsequently,C2iscleavedbytheinitiation complexes yielding a C3 convertase, C4b2b4. In the alternative pathway, C3 proconvertases are formed by binding factor B eithertofluid-phaseC3(HO),aspartoftheso-called‘tick-over’process, or to surface-bound C3b, as the first step in the feed-back amplificationloop.Subsequently,theseproconvertasesarecleavedby solublefactorDyieldingC3convertases,C3(HO)BbandC3bBb5,10,11. Thus,short-livedC3convertasesareformedonsurfaces,providinga short,localizedboostinproductionofC3bmoleculesthatopsonizethetargetedsurfaceforimmuneclearance.Hostcellsareprotected fromopsonizationbyregulatorsthatbindC3borC4btodisruptthebimolecularC3convertasesanddegradeC3bandC4b7,12–14.Dysregulationofthisprotectioncausesarangeofpathologiesoccurringinavarietyofdiseases15–22,whichunderscoresthecriticalroleofopsonizationbytheC3convertases.

C4andC2oftheclassicalandlectinpathwaysandC3andfactorB ofthealternativepathwayarehomologousproteinpairswithaminoacid sequenceidentitiesof30%for human C3 andC4,and 39%for factorBandC2.MatureC3andC4(MWof190and204kDa,respectively) consist of two (β-α) and three (β-α-γ) chains that form 13 domains: eight macroglobulin (MG1-8) domains, a linker, an anaphylatoxin-like (ANA) domain, a ‘C1r/C1s, Uegf, Bmp1’ (CUB) domain, a thioester-containing domain (TED) and a C3, C4 and C5 carboxy-terminal(C345C)domain23–25.Proteolyticremovalofthesmall(9kDa)ANAdomainyieldsC3bandC4b,whichexposetheirthioestermoiety present in TED for covalent surface attachment25,26. C2 and factorBarefive-domainserineproteaseswithMWof92and90kDa, respectively.Astructureofpro-enzymefactorBshowsthatthepro-peptide region forms three ‘complement-control protein’ (CCP) domains and a helix αL in the linker region preceding its von WillebrandtypeA(VWA)domain.InfactorB,pro-peptidehelixαLblocks the conical positioning of C-terminal end of helix α7 of the VWA domain,whichaffectstheorientationoftheC-terminalserineprotease(SP)domain27.Astructureofpro-enzymeC2isnotavailable;however, structuresareavailableforfragmentsC2a(CCP1-CCP2-CCP3)andC2b(VWA-SP)28–30. (We follow the recommendations of Bohlson et al.31, referringtothesmallC2fragmentasC2aandthelargefragmentofC2 asC2b).Structuraldataonproconvertaseandconvertaseformation fromC4andC2,formingC4b2andC4b2brespectively,islimitedto small-angleX-rayscatteringdata32,butisavailableindetailforC3and factor B.Crystalstructures ofthe proconvertaseC3bBand C3bBin complexwithfactorDrevealan‘open’conformationoffactorB,with its scissile loop accessible for proteolytic activation by factor D13. However, small-angle X-ray scattering data32 indicates that pro-convertaseC4b2ismorelikefactorBincomplexwithcobra-venom factor(CVF),whichdisplaysa‘closed’conformationwithaninaccessible scissile bond33. Furthermore, the crystal structures show an extensivebindinginterfacebetweenC3banddomainsCCP2-3offactor B, while interactions with the VWA domain of factor B involve binding of the C3b-carboxy terminus to the ‘metal-ion dependent adhesion site’ (MIDAS) of the VWAdomain. A structure of the convertaseC3bBbisonlyknown in adimericcomplexwithaS.aureus complementinhibitor(SCIN),(C3bBb-SCIN) 34.Intheabsenceofhelix αLofthepro-peptidesegmentBa,VWA-helixα7rearrangesandtheSP domainre-orientsin(C3bBb-SCIN) by142°comparedtoC3bB,while protease fragment Bb is connected to the C-terminal carboxylate moietyofC3bthroughtheMIDASinVWA.Thiscontactpointatthe MIDASisformedbyaveryslowon-offprocess,whichisenabledbythe extensive CCP2-3 contacts in C3bB13. Since the additional contacts provided by CCP2-3 areabsentinBb,reassociationofBbwithC3bis effectively impossible, hence explaining the irreversible anddissociation5,35. By superimposing C3 onto C3b, the dimeric C3bBb-SCINcomplexalsoservesasamodelfortheenzyme-substratecomplex, C3bBb-C3. A dimeric interface formed by MG4-5 domains explainstheinhibitorymodeofcompstatin36.However,thishypotheticalmodelshowsca.30-ÅdistancebetweenthescissileloopinC3and theactivesiteintheBb-SPdomain34.Agapthatmightpresumablybe overcome byflexibilityinthe C345C-neckregionin C3b,allowinga largeswingofBbtowardsthescissileloop.

Here, we present structures of human proconvertase (C4b2), convertase (C4b2b) and convertase-substrate (C4b2b-C3) complex from the classical and lectin pathways of complement activation derivedfromtwocryo-EMsingle-particleexperiments.Proconvertases werepreparedbymixingC4bandC2.Convertasesweregeneratedby mixingC4b,aproteolyticinactivemutantS679AofC2andanactive C1s fragment in the presence of C3. Furthermore, to stabilize the convertase-C3complex,weaddedaduo-nanobodywithalonglinker combining an anti-C3 and anti-C4b nanobody that do not inhibit classical and lectin pathway C3-convertase activity. Single-particle analysisofafirstdatasetresultedindensitymapsofC4b2at3.5-Å resolutionandanalysisofaseconddatasetyieldedmapsofC4b2band C4b2b-C3at4.2and3.5-Åresolution,respectively.

Results
Proconvertasestructure
4662ImageswerecollectedofpurifiedC4b2complexes,resultingina cryo-EMmapata3.5-Åoverallresolution(seeMethods,Table1and SupplementaryFigs.1,2).Thereconstructionrevealedwell-resolved densityformostdomains,exceptforTEDofC4bandSPofC2,which werenotvisibleinthedensity;furthermore,C2-CCP1displayedonly partial density (Fig. 1a). To resolve structural heterogeneity and improvetherepresentationofflexibleregions,thedatasetwasfurther classifiedintotwoparticlesubsets,yieldingmapsat4.0Å(from31,587 particles)and4.2Åresolution(18,613particles)(Table1,Fig.1band Supplementary Fig. 3a). In the 4.0-Å map, additional density correspondingtotheSPdomainbecameapparent,althoughatlowlocal resolution(~5.5–7.5Å).The4.2-Åmaprevealedaveryweakdensityfor theSPdomain,thatwasspatiallydisplaced(SupplementaryFig.3a); however,weomittedthisdomainfromthemodel,becauseitsorientationcouldnotbeestablished.

TheconformationofC4bintheproconvertasestructurewaslike thatofthestructureofC4b,exceptforahighlyvariableorientationof TED and a rotation by 29° of C345C, consistent with variations in C345CorientationobservedinotherstructuresofC4b(SupplementaryFig.3b)25,37,38.

TheinitialmodelforC2wasderivedfrompreviouslypublished structuresofC2aandC2b(previouslyreportedascrystalstructuresof C2bandC2a,respectively)28–30.Usingboththe3.5-Åand4.0-Åmaps, residues 209-233 of the CCP3-VWA linker were built; however, the scissileloop(residues234–250)remainedunresolved.VWA-helixα7 (res.442-454)wasremodelledandadditionaldensityinthe4.0-Åmap allowedthebuildingoftheVWA-SPlinker(res.455–462),andpositioningoftheSPdomain(res.463-752).Thus,themodelofC2inthe proconvertaseincludesdomainsCCP1-3,linker,VWAandSPdomain, butlacksthescissileloop(res.234-249)andtheC4b-TEDdomain. CCP2-3 in C4b2 are arranged as in the C2a crystal structure30, whileCCP1isrotatedby11°.ThelinkerhelixαL(res.213-233)docks intoagrooveoftheVWAdomainandtheC-terminalendofVWA-helix α7(res.451-454)appearsdislocated,whilsttheN-terminalpartofhelix α7(res.442-446)ispositionedinitscanonicalgroove,similartothat seeninthestructuresoffactorBandC3bB13,27.ThepositionofC2SPin the4.0-ÅresolutionmapresemblesthatoftheSPdomainofC3bBin theopenconformation(Fig.1b,SupplementaryFig.3c).Moreover,in C2,theP1residue(Arg243)ofthescissilebondisnotboundbylinker- helixαLandVWA-helixα7;andlikeinC3bB,thescissileloopisflexible andaccessibleincontrasttoaboundandinaccessibleArg259infree factorBandinCVF-B(SupplementaryFig.3d)33.However,theposition oftheSPdomaininC4b2ishighlyvariable,asindicatedbythepoor density;thus,suggestingadynamicequilibrium,similartowhathas beensuggestedforC3bB39.

ContactsbetweenC2andC4bintheproconvertaseareobserved between the C2-CCP1and C4b-C345Cdomains, between C2 CCP2-3 andC4bMG2,MG6-7andCUB,andbetweenC2VWAandC4bC345C (Supplementary Fig. 4a). Extensive amino-acid interactions that are welldefinedbythedensityareobservedattheC2CCP2-3site(Supplementary Fig. 5a). C2 CCP2 interacts with residues of the C4b (cleavedα)α’chainN-terminalregion(α’NT)thatliesoverC4bMG7 andwithresiduesofMG6,whereasseveralresiduesofC2CCP3 interactwithresiduesofC4bMG7andCUB.Aminorcontactareais formedbetweenC2CCP1andC4bC345C.Finally,theMIDASregionof C2 VWA contacts the C-terminal end of C4b C345C, where the C-terminusofC4bisexpectedtobindintotheMIDAS.However,the density in this area is too poor for detailed interpretation (SupplementaryFig.5b).

Convertasestructure
Inoneexperiment,convertasesandconvertase-substratecomplexes were formed, by mixing C3-C4b complexes linked by the duo-nanobody with C2 S679A and preactivated C1s in the presence of 2mM Mg2+ (see Methods). 4276 Images were collected from this sample yielding 17,073 particles. A density map at 4.2-Å resolution showedtheconvertaseC4b2bwithduo-nanobodysegmentNbC4bB12 attached(Fig.2,Table1,SupplementaryFigs.6–8).C4bandNbC4bB12 showeddensityconsistentwiththeoverallmapresolution,however, theC2domainsVWAandespeciallytheSPyieldedsignificantlylower localresolution(Fig.2a).AlldomainsofC2bandC4bandNbC4bB12 wererigidbodyfittedwithminimalfurtheradjustmentandrefinement (Fig.2a),see“Methods”.

ThestructureofconvertaseC4b2brevealedC4binitscommon conformation25,32, while the density for C2b was insufficient for detailedinterpretation.BindingoftheNbC4bB12segmentoftheduo-nanobody to C4b was identical to what was observed previously38.
Giventhehighermapresolutionobservedfortheconvertase-substrate C4b2b-C3complex,structuralchangesfromproconvertaseC4b2to convertase C4b2b will be discussed in detail later; instead here we describetheoverallchangesindomainpositionsonly.

Removalofthepro-peptidesegmentC2a(CCP1-3andαL)from theproconvertaseC4b2yieldsaseriesofdisplacementsofC4bC345C, C2bVWAandC2bSPinC4b2b(Fig.2b).IntheabsenceofC2a,C2b VWArotatesby34°withrespecttothemainbodyofC4band25°with respecttotheC345CdomainofC4b,whileC4bC345Crotatesby22° (SupplementaryFig.9a).Inthisnewposition,C2bVWAmakescontacts withtheCUBdomainofC4b(Fig.2b).ThisinvolvesresiduesArg1336 andAsn1337ofC4bCUBpossiblyinteractingwithresiduesGln365and Asn397ofC2bVWA,whichwerepreviouslyinvolvedininteractions with C2 CCP3 in the proconvertase (Supplementary Fig. 9b). The removal of αL altersthe VWA-SP interface, and the C2b SP domain rotatesby124°(SupplementaryFig.9c)relativetoVWA.Theoverall resultofthesedomainrotationsisashiftof33Å(inS679ACαposition) of the SP active site and reorientation by 142° of the SP substrate-bindinggroove(Fig.2b).

Convertase-substratecomplex
FormationofC4b2b-C3wasfacilitatedbymutatingthecatalyticresidueS679AofC2andbystabilizingtheconvertase-substrateinteractionthroughadditionofanti-C3hC3Nb3andanti-C4bNbC4bB12duo-nanobody.177,801particles,fromthesamedatasetasthatusedfor theconvertasestructure,revealedC4b2b-C3complexesatanoverall resolutionof3.5Å(Table1,SupplementaryFig.7,8).C4b,C2b,C3and anti-C4 NbC4bB12 were docked into the density. All domains were rigid-body fitted, followed by manual fitting and real- space refinement.Thedensitywaswellresolvedforalldomains,exceptforC4b CUBandTED,C3C345CandtheC-terminalpartofNbC4bB12(Fig.3a). Furthermore, no density was observed for the anti-C3 nanobody hC3Nb3,whichbindstothetopoftheC345CofC3,consistentwithits poorly defined density in negative-stain images of C3-hC3Nb3 complexes40. Binding of the anti-C4b NbC4bB12 was identical to whatwasobservedinthe convertaseandaspublishedpreviously38. Modellinganti-C3hC3Nb340ontotheC345Cyieldeda94-Ådistance betweenN-andC-terminusofthenanobodymodels(Supplementary Fig.10a),whichwaseasilyaccommodatedbythedesignedlinkerwith anestimatedlengthof177Å.

ThedensitymapofC4b2b-C3showedthecriticalC2b-C4bcontactoftheconvertase,betweenC2bVWAandC4bC345C,atalocal resolutionof4–5Å.Thedensityisconsistentwithacarboxylatemoiety oftheC4b-γchainchelatingtheMg2+ionoftheMIDASinC2bVWA (SupplementaryFig.10b).TheorientationsofC2b-VWAdomainswith respecttoC4bC345Cdifferbyupto38°inthestructuresofC4b2, C4b2bandC4b2b-C3(SupplementaryFig. 10c). This variabilitysuggeststhattheregularprotein-proteininteractionsareweakandsubsidiarytothepivotalinteractionofthecarboxylatemoiety(chelating the Mg2+) that sits at the end of a very short (2 residues) flexible C-terminaltail.The flexibilityatthisinterface is consistentwith the lowerlocalresolutionsobservedatthisinteractionsite.

The density map of C4b2b-C3 resolves VWA-helix α7 and the subsequent VWA-SP interface of C2b (Supplementary Fig. 10d, e). Upon removal of αL of the pro-peptide in C4b2, VWA-helix α7 readjusts (residues 442-452), shifting by approximately one helical turn(Fig.3b).HydrophobicinteractionsbetweenVal413(loopβEα6), Leu439(loopβFα7),andLeu445(helixα7)arepreserved,resultingina concerted movement (Fig. 3c). This causes a displacement of loop βEα6whichliesinbetweenα7andtheMIDASsite(Fig.3d).Comparable changes occur in integrin-I domains41,42, when forming a high affinity MIDAS state, with loops βAα1, βDα5 and βEα6 adopting equivalent conformations (Supplementary Fig. 10f). However, in (C3bBb-SCIN) loop βFα7 of Bb (including Val430, equivalent to 2 Leu439in C2b) is affected due to contacts with SCIN, breaking the contactsbetweenthethreehydrophobicresidues(Val430,Val455and Val464)leavingthepositionofloopβEα6almostunchangedincomparisontoC3bB(SupplementaryFig.10g).TheC-terminalpartofC2-VWAhelixα7(res.451-454)movesintothepositionpreviouslyoccupiedbyresidues221-226ofαLhelixandnowinteractwiththenewlyformedN-terminusofC2b(Fig.3b).ThesechangesinVWAaffectthe linker453-462thatdominatestheVWA-SPinterface(Supplementary Fig.10e).TheVWA-SParrangementobservedintheC4b2b-C3complex isidenticaltothatobservedinstructuresoffreeC2b,indicatingthe stabilityofthisVWA-SParrangement.

Binding of C3 to the convertase C4b2b introduces changes in C4b2bandC3(Fig.3e,f).TheC345CdomainofC4brotates15°and bothVWAandSPdomainofC2brotateby19°.VWAandSProtateas oneby13°withrespecttoC345C(SupplementaryFig.10h).Overall, these motions shift the SP active site Cα of residue S679A by 29Å towardsthescissileloopofboundC3(Fig.3e).ThestructureofC3 changesby9°inα-βchainorientationfromthatoffreeC3(Fig.3f)23to form two C4b-interaction sites that allows contacts with C2b and dockingofthescissileloopintheproteaseactivesite(Fig.3g).

OneC4b2b-C3contactsiteisformedbytheMG4-5domainsofC3 andC4b(Fig.4a);thisinteractionsiteisequivalenttothedimericMG4- 5 site observed between two C3b molecules in (C3bBb-SCIN) 34 (Supplementary Fig. 11a). The arrangement of MG4-5 of bound C3 differsby11–12°fromthatoffreeC3,C3band(SCIN-C3bBb) (SupplementaryFig.11b).TheC4b-C3MG4-5interfaceburies1309Å2and involvesaseriesofhydrophobiccontactssupportedbysurrounding polarandchargedinteractions(Fig.4b).

AsecondinterfaceisdominatedbytheMG7domainsofC3and C4b, with additional contacts provided by MG3 and MG6 of both molecules,covering890Å2(SupplementaryFig.5c).Asetofdomain rearrangementsinC3allowformationofthissecondinterface.MG3,6 and7rotateby33°,5°and15°,respectively(superposingunboundand convertase-boundC3ontheirMG5domains)(SupplementaryFig.11c).In consequence, His918 atthe tip of MG7 shifts 6.5Å towards C4b, whereC3loop917-920interactsextensivelywithC4bresiduesofMG6, MG7andα’NT(Fig.4c).Moreover,C3MG6formselectrostaticinteractionswithC4b-MG3andMG6andchargedresiduesofC3MG3and MG7provideadditionalinteractionswithC4bMG6residues,further stabilizingtheMG3,6-7contacts(SupplementaryFig.11d).

FormationofthetwoC3-C4binteractionsitesallowC3domains MG3 and ANA (after 33° and 10° rotations, respectively, relative to MG5)tocontacttheC2b-SPdomain(SupplementaryFig.11c).Overall, theconformationalchangesinC3allowthescissilelooptoshiftby6Å (inCαpositionofVal741)anddockintothesubstate-bindinggroove (Fig.4d).InternalH-bondsbetweenHis743-His738andSer672-Asp752 andinteractionswithsubstate-bindinggrooveyieldanorderedscissile loop,whereasresidues742-750weredisorderedinthemodeloffree C3(Fig.4e).TheMG3domainofC3interactswithresiduesofloopA (Lys485),loopB(Leu517,Arg519)andloopE(Trp529)oftheC2bSP domain(Fig.4f).ComparedtofreeC2b(PDB2I6Q)28,loopA,Band3 rearrange to allow access and binding of the scissile loop into the substratebindinggroove(Fig.4g);whereas,inasecondstructureof free C2b (PDB 2ODP)29 loops A and B are disordered, while loop 3 resemblesalreadytheboundstate(SupplementaryFig.11e).InBbof (C3bBb-SCIN) , the equivalent loops resemble the unbound state 2 (SupplementaryFig.11e).Theprotease,C4b2b,bindsthescissileloop in the substrate-binding groove with Leu746, Arg748 and Ser749 dockedintotheS3,S1andS1’substrate-bindingpockets(Fig.4h).The C2bcatalyticsitetriad(His507,Asp561andS679A)andoxyanionhole, stabilizedbytheguanidiumgroupofArg714,adoptarrangements compatible with catalytic activity, whereasthe oxyanion hole isnot fullyformedinthestructuresoffreeC2bnoranyoftheBbstructures.

Overall,thestructureofC4b2b-C3resemblesthatofC3bBb-C3b takenfrom(C3bBb-SCIN) (SupplementaryFig.11f).ConvertaseC3b 2 andC4barearrangedsimilarly.ThepositionofproteasefragmentBb liesinbetweenthatofC2binC4b2bandC4b2b-C3,respectively(differingbyrotationsof8°and13°,SupplementaryFig.11g),heldinplace bythetwoSCINmoleculesbridgingthedimericpairofC3bBbcomplexes. As predicted previously, C3b-C3b interactions in the SCIN-inhibited C3bBb dimer mimicin partC3 binding tothe convertase. However,C3bcontactstheconvertase(C3bBb)onlythroughtheMG4- 5 domains, while the two C3b-MG7 domains are ca. 14Å apart in (C3bBb-SCIN) . In comparison, C3 MG7 of C4b2b-C3 and the corre- 2 sponding C3b MG7 of (C3bBb-SCIN) differ by 40° in orientation. 2 Moreover,C3bMG7bindsthe negativelychargedα’NTtailontoits surface.Altogether,whileinC4bC2b-C3apositivelychargedsurfaceof C3MG7facesanegativelychargedsurfaceofC4bMG7andα’NT,in (C3bBb-SCIN) two negatively charged surfaces of MG7 and α’NT 2 opposeeachother(Fig.5).

Methods
C4b2complexformationandpurification
Similar to Mortensen et al.25,32, we used C2 and C4b derived from pooledhumanserum(thuscontainingamixtureofC4AandC4Bisotypes)(ComplementTechnology,Inc.)Bothproteinsweremixedat finalconcentrationsof3.2and2.1μM,respectively,in20mMHEPES pH7.4,150mMNaCl,3mMMgCl ,supplemented with 0.2% glutar- 2 aldehydeforcrosslinking.Themixturewasincubatedfor30min.on ice and quenched with 1M Tris pH 8.0 to a final concentration of 100mM.TheC4b2complexwaspurifiedviasizeexclusionchromatography (SEC) on a Superdex 200 Increase 10/300 column pre-equilibratedinPBSandkeptoniceuntilcryo-EMgridpreparation.

C4b2b-C3complexformationandpurification
C3waspurifiedfromhumanserumbydifferentialpolyethyleneglycol precipitationtoenrichcomplementcomponents,followedbyanion-exchangechromatographyonaDEAEmatrixtoisolatenativeC3.Final polishingstepsremovedresidualserumproteinsandyieldedhomogeneous,functionalC323.Previouslygeneratedconstructsforinactive C2(S679A)andaC1sfragmentconsistingofdomainsCCP1,CCP2and theSPdomain(res.293-688)withthenativecleavagesiteatresidues 433-438substitutedwithanenterokinasecleavagesite(DDDDK)were used.N-terminalhexahistidinetaggedC2(S679A)andC1s,weretransiently expressed in HEK293-E+ and HEK293-ES cells, respectively (Immunoprecise Antibodies BV) and harvested 6 days post-transfection. A duo-nanobody construct was engineered by fusing anti-C4b38 and anti-C3 nanobody hC3Nb340 separated by a linker containing a non-complement specific nanobody 4C848 flanked by thrombincleavagesitesfollowedby3xGSSrepeats.Theconstructwas cloned into a modified pET13 vector encoding a C-terminal hexahistidinetagandexpressedinE.coliBL21(DE3)pLysS.Hexahistidine-tagged proteins were purified using Ni-Sepharose for the duo-nanobody and Ni-Sepharose Excel beads for C2 (S679A) and C1s (DDDDK)(GEHealthcare)at4°C,usingstandardprotocolsandafurtherSECstepusingaSuperdex7510/300column(GEHealthcare)preequilibrated in 20mM HEPES pH 7.4, 150mM NaCl. Peak protein fractionswerepooled,analyzedbySDS-PAGEgeltoconfirmthepresenceofallproteincomponents,andstoredat4°Cforupto3days priorcryo-EMgridpreparation.

25μg of C1s (DDDDK) at an initial concentration of 28μM was activatedbycleavagewith16unitsenterokinase(NewEnglandBiolabs Inc,P8070L)at37°Cfor1hinatotalvolumeof21μLin20mMHEPES pH 7.4, 150mMNaCl buffer containing 2mM CaCl . Human serum- 2 derived C4b (Complement Technology, Inc), C3, and the duo-nanobody were mixed to a final concentration of 5μM each. The C4b-C3duo-nanobodycomplexwaspurifiedbySECusingaSuperdex 200Increase3.2/300columnpre-equilibratedin20mMHEPESpH7.4, 150mM NaCl, 2mM MgCl . The C4b-C3 duo-nanobody complex 2 (0.20mg/ml), C2 (S679A) (0.50mg/ml) and activated C1s(DDDDK) (0.05mg/ml)weremixedina25:5:1volumeratio,incubatedfor30sec at room temperature and immediately used for cryo-EM grid preparation.

Cryo-EMgridpreparationanddatacollection
3μlofC4b2(0.20mg/ml)orC4b2b-C3(0.23mg/ml),wereappliedtoa glowdischargedR1.2/1.3200meshAuholeycarbongrid(Quantifoil) andplunge-frozeninliquidethaneusingaVitrobotMarkIV(Thermo FisherScientific)at4°Cand22°C,respectively.

C4b2cryo-EMdatawerecollectedona300kVTitanKriostransmissionelectronmicroscope(ThermoFisherScientific)equippedwith aGatanK3SummitdirectelectrondetectorandaGatanBioQuantum post-columnenergyfilteroperatedwitha20eVslitwidth.Datawere acquired in counted super-resolution mode using EPU-2.10.00 (Thermo Fisher Scientific) at a nominal magnification of 105,000×, correspondingtoasuper-resolutionpixelsizeof0.418Å/pixel.4662 movieswerecollectedusingadefocusrangeof−1.0to−2.2μm,ata totaldoseof50e−/Å2across50frameswith2.2sexposuretime.

C4b2b-C3cryo-EMdatawereacquiredusinga200kVTalosArctica transmission electron microscope (Thermo Fisher Scientific) equipped with a K2 Summit direct electron detector (Gatan) and a post-columnGIFQuantumenergyfilter(Gatan)operatingwitha20eV slit width. Two datasets were collected from the same specimen in counting mode using EPU-2.9.00 (Thermo Fisher Scientific) at a nominal magnification of 130,000×, corresponding to a calibrated pixel size of 1.04Å atthe specimen level. A totalof 1700 and 2576 movieswerecollectedforthetwodatasets,respectively,overadefocusrangeof−0.8to−2.6μm.Eachmovieconsistedof40frames.The first dataset was recorded with a total dose of 52 e−/Å2 and a total exposuretimeof4.0seconds,andthesecondwithatotaldoseof50 e−/Å2over6.0seconds.

ProcessingofC4b2cryo-EMdata
Cryo-EMdataforC4b2wereprocessedusingCryosparcv3.2/349.The datasetwascollectedinsuperresolutionmode(0.418Å/pixel),itwas importedand2×binnedtoapixelsizeof0.836Å/pixel,followedby patch- based motion correction, patch-based CTF estimation, and moviecuration.Aftercuration,4486movieswereretainedforfurther processing.Initialblobpickingwasperformedonallcuratedmicrographs,followedbyparticleextractionusingaboxsizeof480pixels, binnedto256pixels.Threeiterativeroundsof2Dclassificationand particleselectionyielded765,438particles.Theseparticlesweresubjectedtoabinitioreconstructiontogeneratethreeinitialmodels(one high-qualitymodelandtwodecoys).Theresultingmodelswereused in several rounds of heterogeneous refinement, followed by non-uniform refinement, yielding an improved 3D reconstruction from 190,374 particles. To improve the resolution of the VWA and SP domains,amaskcoveringC2andtheC4b-C345Cdomainwasapplied totherefinedvolume,andthedataweresubjectedto3Dvariability analysis, producing four distinct clusters. These clusters underwent heterogeneous refinement, and the most well-resolved cluster contained111,565particles.Theseparticleswerere-extractedattheoriginal box size (0.836Å/pixel), and subjected to non-uniform refinement, producing a 3.5-Åmap, according to the goldstandard FSC=0.143criterion.ThismapshowednodensityfortheC4b-TEDand theC2-SPdomain,andonlypartialdensityforC4bCCP1.

Further 3D-variability analysis and iterative heterogeneous refinementofthe111,565particlesresultedintwomapsthatrevealed theC2-SPdomainindistinctorientations.Onecluster,composedof 30,885particles,yieldedthebest-definedfeaturesofC2.Thesecond, from18,613particles,produceda4.2-Åmapwithweakdensityforthe SPdomain.Thebest-definedC4b2mapwasthenreprocessedthrough multipleroundsof3D-variabilityanalysisand heterogeneous refinement, startingagainfromthe190,374particles.Thefinalnon-uniform refinementyieldeda4.0-Åresolutionmap,basedon31,587particles. ThisreconstructionrevealedtheC2-SPdomainatalocalresolutionof ~5.5–7.5Å(seeTable1).

ProcessingofC4b2b-C3data
TwodatasetsoftheC4b2b-C3complexwereprocessedinCryoSPARC v4.2.0/4withoutbinning.Eachdatasetunderwentpatch-basedmotion correctionandCTFestimation,followedbymoviecuration,resulting intheselectionof1384and1906movies,respectively.Aninitialsubset of100movieswasusedforblobpicking,generating2Dtemplatesthat guidedparticlepickingacrossthefulldatasets.Atotalof448,154and 615,097particleswereextractedusingaboxsizeof380pixels,Fourier-croppedto256pixels,andsubjectedtomultipleroundsofindependent 2D classification. Selected 2D classes from both datasets were mergedandsubmittedtoanadditionalroundof2Dclassificationand selection, yielding a final pool of 589,457 particles. These particles underwentmultipleroundsofheterogeneousrefinementusingdecoy models, followed by non-uniform refinement, resulting in a reconstructionoftheC4b2b-C3complexat3.5 Åresolution(gold-standard FSC=0.143) from 177,801 particles. These were re-extracted at the originalboxsizepriortothefinalrefinement.

OneofthedecoymodelsrevealedclearfeaturesofC4balongwith partiallyresolveddensityforC2b,correspondingtotheC4b2bcomplex.Thismapwasfurtherprocessedfollowingthesamestrategyused previouslytoresolvetheSPdomaininC4b2.AmaskcoveringtheC4b-C345CdomainandC2bwascreated,andthemapalongwithitscorresponding particles,wassubjected to multiple rounds of3Dvariability analysis and iterative heterogeneous refinement, ultimately improvingthefeaturesofC2b.Fromthissubset,17,073particleswere re-extractedandrefined,yieldinga4.2Åresolutionmap.

Modelbuildingandrefinement
TheC4b2modelwasgeneratedbydockingthecrystalstructuresof C4b(PDB5JTW)50,C2b(PDB2I6Q)28,andC2a(PDB3ERB)30intothe 3.5Å cryo-EM density map using domain-by-domain fitting in ChimeraX51. Rigid-body refinement was performed in Phenix52, followedbymanualrebuildinginregionswheredensitydeviatedfrom theinitialmodels.Iterativecyclesofmodelbuildingandlocalrefinement were conducted in Coot53 and Phenix. Several regions were omittedfromthefinalmodelduetoinsufficientdensity,includingthe C4b-TED domain (residues 985–1325), residue 757 of the C4b N-terminalα-chain,residues1391–1392linkingC4b-CUBtoMG8,and the C2- SP domain (residues 455–752). The C2-CCP1 domain was refinedbyrigid-bodyfittingonly,withresidues74–80excludeddueto poordensity.DensityallowedmodelingofthefirstN-glycanatC4b residuesN226,N862,andN1328,aswellasthemodellingofC2helixαL (residues209–229)andrebuildingofC2helixα7(residues444–454). Thescissileloop(residues230–250)wasunresolvedandthereforenot built. The C-terminal tail of the C4b α’-chain (residues 1414–1421), absentintheoriginalcrystalstructure,wasresolvedandbuiltdenovo. ThefinalmodelwasrefinedinrealspaceusingPhenixwithstandard geometricrestraints.

Forthe3.9Åcryo-EMmap,whichcontainedadditionaldensityfor theC2-SPdomain(residues463–752),therefinedC4b2modelserved asastartingpoint.TheSPdomain(PDB2I6Q)28,withoutglycans,was rigidbodyfittedinChimeraXandrefinedinPhenix.Thelinkerbetween theC2-VWAandC2-SPdomains(residues455–462)wasmanuallybuilt inCoot.Densitysupportedmodelingofresidues231–233inthescissile loop, while residues 234–250 remained unresolved. Refinement followed the same protocol as above, with the C2-CCP1 and C2-SP domains kept fixed after rigid-body fitting. This yielded a lower-resolutionC4b2modelincorporatingtheSPdomain.

TogeneratetheC4b2bmodel,thestructureofC4bincomplex withNbC4bB12(PDB7B2Q)38wasdockedintothe4.2Åcryo-EMmap. AsthismodellackedtheC4b-C345Cdomain,residues1594–1744from theC4bcrystalstructure(PDB5JTW)wereused.Giventherelatively lowresolutionofthemap,allC4bdomains,NbC4bB12,andC2b(PDB 2I6Q)wererigidbodyfittedinChimeraXandrefinedinPhenix.Limited manualbuildingwas performedinCoot,includingextensionofthe C4b N-terminal α’-chain (residues 762–767) and repositioning of sidechainsofresiduesArg1336andAsn1337.ResidueSer679inC2b wasmutatedtoalanine,reflectingtheconstructusedforsamplepreparation.ThefirstN-glycanwasretainedoraddedatglycosylationsites N226,N862,N1328,andN1391ofC4b,andatN290,N467,andN470of C2bbasedonmapdensity.

The C4b2b-C3 model was built using the same approach as for C4b2b, using the C2b structure (PDB 2ODP)29 and correcting pointmutationsaccording totheexperimentalconstruct(A261C revertedtowild-typeandS679Aintroduced).C3(PDB2A73)23was alsoincorporatedintothemodel.Initialdomain-by-domainrigid-body fitting was followed by iterative manual rebuilding in Coot and real-space refinement in Phenix with geometric restraints. The3.5ÅmapallowedmodelingofproteinC3N-terminalα-chain residue672andscissileloopresidues742–750.LoopregionsinA, B and 2 of C2b (residues 487–490, 509–517, and 707–711, respectively)werebuilt.N-glycansweremodeledatC2bresidues N290,N467,N471,and N621,andatresiduesN226andN862for C4b. For protein C3, residues 74–75 and 312–313 were modeled, while residues 102 and 1497–1499 were omitted due to lack of density. The first N-glycan was also added at residues N85 and N939 where density permitted it and refined as previously described.

Illustrations portraying protein structures were prepared using Pymol(Schrödinger)andUCSFChimeraX51.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The is an important manuscript that reports the Cryo-EM structures of the complement classical / lectin pathway C3\nproconvertase, C3 convertase and C3 convertase with its substrate C3. These structures provide significant information\nabout the structural or conformational changes of the activated components during the complement activation processes.\nThat information would be relevant for drug designs of medical intervention controlling immune effector functions and\nameliorating inflammatory response to reduce complement-mediated tissue injuries. There are two important issues that are\ndesirable to be addressed.\nFirst, the source of C4 or C4b used was only vaguely described (line 380). Were “serum-derived C4b and C2 originated from\na single human subject or from mixed sera of multiple human subjects. This is particularly relevant for human C4 because of\nits extensive polymorphism of C4A and C4B isotypes with multiple allotypes in each protein. The polymorphic sites in C4\nare overwhelmingly clustered at the TED domain and most human subjects consist of both acidic and basic isotypes.\nUnfortunately, cryoEM structures of the TED domain were not resolvable in this manuscript and thus their structural and\nfunctional relevance were obscure. In retrospect, the use of source proteins from single human subjects with minimal\nvariations would have yielded more definitive or informative structures.\nSecond, dis-similar to many colleagues including the same senior author of this manuscript who published on complement\nstructures, the heavy chain of C2 with serine proteinase and vWA domains (after activation0 was referred to as C2b instead\nof C2a. This creates unnecessary confusions, and feelings to many colleagues. Irrespective to the reasons of the\nnomenclature switch, it would be appropriate to add a footnote explaining the change of nomenclatures, or use a new set of\nnames for the heavy and light chains of activated C2. The authors could acknowledge the limitations of using the reversed\nnames for C2a and C2b in the Discussion", "reviewer_2": "The paper by De la O Becerra et al presents Cryo EM structures of the classical C3 proconvertase, convertase and the\nconvertase-substrate complex. A number of novel insights are provided as to the similarity of the classical convertase to the\npreviously described alternative convertase as well as the mechanism of classical convertase activity and formation. The\nstructure of the convertase-C3 complex is very interesting and resolves questions related to how interaction of the protease\ndomain with the scissile bond is achieved. Previous work with the C3bBb-SCIN complex suggested a general explanation\nof the mode of C3 binding, especially the importance of the MG4-5 domains of both components but also indicated that there\nmust be more to the story. The structure of the classical convertase-C3 structure answers the remaining questions showing\nthat a number of relatively small structural movements combine create additional converatse-C3 interactions to form a clear\nenzyme-substrate complex. Apparently, the absence of the SCIN inhibitor also allowed explanation of the increased strength\nof the VWA MIDAS-C4b (and presumably C3b) interaction after activation of the protease component. The structures in the\npaper are of high quality with poorly resolved regions being restricted to areas of less relevance to the study.\nThe complex with the substrate produced a very good structure. The scissile bond loop is very well resolved as is the newly\ndescribed contact site involving the MG7, MG6 and MG3 domains. The chromatogram in figure S6 seems to suggest that the\ncomplex was probably stable enough to be analyzed without the linker peptide.\nThe paper is well written and the figures are of good quality. Overall, I don't think the paper requires any real modification.", "reviewer_3": "This interesting manuscript tackles one of the unsolved structural problems in the complement field, the assembly of the\nclassical pathway C3 convertase. As such it is of some importance - particularly in the context of evolving therapeutic\napproaches to inhibiting complement activation.\nOne general concern is the decision to call the convertase C4b2b. The convertase was, from its first description in the\n1960s, termed C4b2a. In recent years there has been a move from some quarters to \"rationalise\" by referring to the\nconvertase as C4b2b; as the authors know, this is an area of continued disagreement in the field. This should be noted in\nthe Introduction and the reason for deciding on C4b2b in the current Ms stated.\nThe authors are very expert complement structural biologists and I cannot criticise their cryoEM analyses and interpretation;\nhowever, I do have some concerns that should be addressed.\nMy major concern is around the generation of the complexes for analysis.\nThe C4b2 complex is generated by simply mixing commercially sourced plasma-derived C4b and C2 at 2.1 and 3.2 um\nrespectively in the presence of glutaraldehyde to non-specifically cross-link. How was this method arrived at and what is the\nevidence that the complex generated in this artificial way represents the labile proconvertase? Indeed, the gels shown in\nsupp Fig 1 suggest that there is very little C2 in the isolated final complex!\nConversion of proconvertase to convertase is mediated (I think - not clear in methods) by incubation of proconvertase with\n\"preactivated C1s\" but evidence of cleavage is not provided.\nTo generate convertase-substrate complexes, C3-C4b complexes are first generated by mixing the two proteins together\nwith a cross-linking nanobody. Again, what is the evidence that this artificially generated complex adopts a conformation\nrelevant to the natural convertase? This complex is then incubated with inactivated C2 and preactivated C1s to yield a\nnanobody-stabilised C4b2bC3 complex. once more, what is the evidence that this complex is representative of the native\ncomplex? supplementary figure 6 shows gels of the C3-C4b-nb complex but not of the complex incorporating C2b, essential\nfor the characterisation.\nMinor points:\nFigure legends are brief and would benefit from more explanation for non-experts;\nSome annoying abbreviations - for example, line 68 and elsewhere - \"resp.\" for respectively, should be eliminated."}, "author_response": "Point-by-point reply\nReviewer #1:\n1.1 First, the source of C4 or C4b used was only vaguely described (line 380). Were\n“serum-derived C4b and C2 originated from a single human subject or from mixed\nsera of multiple human subjects. This is particularly relevant for human C4 because\nof its extensive polymorphism of C4A and C4B isotypes with multiple allotypes in\neach protein. The polymorphic sites in C4 are overwhelmingly clustered at the TED\ndomain and most human subjects consist of both acidic and basic isotypes.\nUnfortunately, cryoEM structures of the TED domain were not resolvable in this\nmanuscript and thus their structural and functional relevance were obscure. In\nretrospect, the use of source proteins from single human subjects with minimal\nvariations would have yielded more definitive or informative structures.\nWe purchased C4b from Complement Technology Inc., as indicated in line 390, which\nis indeed a mixture of C4A and C4B. A mixture of C4A and C4B obtained from pooled\nplasma is commonly used in structural studies (see [25, 32, 37, 38, 48]). Also, the set-\nup of this study was not focused on differentiating C4A and C4B. Moreover, differences\nin C4A and C4B localize to exposed areas in TED that do not form contact with other\nparts of the complex. Flexibility of the TED domain was anticipated, because variability\nin C3b-TED positioning was observed in the convertase structure of (C3bBb-SCIN)\n2\n(as shown by the very high B-factors). Most likely, the poor resolution is this area is\nforemost due to lack of interactions of TED, causing variations in its position, and to a\nlesser extent due to differences in C4Ab and C4Bb.\nWe added at line 393-396:\n“Similar to Mortensen, et al. (2015 and 2016), we used C4b and C2 derived from pooled\nhuman serum (thus containing C4A and C4B isotypes) (Complement Technology, Inc.)…”\n1.2 Second, dis-similar to many colleagues including the same senior author of this\nmanuscript who published on complement structures, the heavy chain of C2 with\nserine proteinase and vWA domains (after activation was referred to as C2b instead\nof C2a. This creates unnecessary confusions, and feelings to many colleagues.\nIrrespective to the reasons of the nomenclature switch, it would be appropriate to add\na footnote explaining the change of nomenclatures, or use a new set of names for the\nheavy and light chains of activated C2. The authors could acknowledge the limitations\nof using the reversed names for C2a and C2b in the Discussion\nWe follow the recommendations of Bohlson, Garred, Kemper and Tenner (2019).\nOriginally, the large enzymatic fragment of C2 was called ‘C2a’ for ‘activated C2’;\nwith\nthe small fragment called ‘C2b’. For all other complement components, including the\nhomologous factor B, ‘a’ refers to the small fragment and ‘b’ to the large fragment.\nNaming the large C2 fragment ‘C2b’ improves the consistency in nomenclature, in\nparticular with respect to C2 and factor B. Unfortunately, the reference to Bohlson et\nal. was missing in the manuscript.\nWe added at line 68:\n“(We follow the recommendations of Bohlson et al. [31], referring to the small C2\nfragment as C2a and to the large C2 fragment as C2b.)”\nWe changed “… activation. These structures, …” at line 301 into:\n“… activation. Following the recommendations of Bohlson et al. (2019) [31], we refer\nto the large fragment of C2 as C2b, and not C2a (which originally indicated ‘activated’\nfragment of C2). Hence, the fragments C2a and Ba vs. C2b and Bb of homologous\nproteases C2 and factor B refer to the equivalent parts. The structures of C4b2, C4b2b\nand C3-C4b2b, …”\nAt line 129 we added after C2b:\n“(previously reported as crystal structures of C2b and C2a, respectively)”\nReviewer #2:\nThe paper is well written and the figures are of good quality. Overall, I don't think the\npaper requires any real modification.\nNo modifications needed.\nReviewer #3 (Remarks to the Author):\n3.1 One general concern is the decision to call the convertase C4b2b. The\nconvertase was, from its first description in the 1960s, termed C4b2a. In recent years\nthere has been a move from some quarters to \"rationalise\" by referring to the\nconvertase as C4b2b; as the authors know, this is an area of continued\ndisagreement in the field. This should be noted in the Introduction and the reason for\ndeciding on C4b2b in the current Ms stated.\nSee reply 1.1\n3.2 My major concern is around the generation of the complexes for analysis.\nThe C4b2 complex is generated by simply mixing commercially sourced plasma-\nderived C4b and C2 at 2.1 and 3.2 um respectively in the presence of glutaraldehyde\nto non-specifically cross-link. How was this method arrived at and what is the\nevidence that the complex generated in this artificial way represents the labile\nproconvertase? Indeed, the gels shown in supp Fig 1 suggest that there is very little\nC2 in the isolated final complex!\nIn fact, the method of complex formation of C4b2 is a, straightforward, solution\nequivalent of that what is happening on a targeted surface. Mortensen, et al. (2016) [32],\ngenerated C4b2 complexes in a similar way (though they stabilized the complex using\nNi2+ (instead of Mg2+) and did not use a crosslinking molecule).\nIndeed, the efficiency of complex formation was low (in line with a low amount of C2\non the gel in suppl fig. 1). In the EM analysis we found particles containing C4b + C2\nbesides many particles consisting of C4b only. The generated C4b2 complex was in\noverall organization consistent with the structural data obtained for the homologous\nC3bB.\nWe added at line 393-396:\n“Similar to Mortensen, et al. (2015 and 2016)[25,32], C4b derived from pooled human serum\n(thus containing C4A and C4B isotypes) and C2 (Complement Technology, Inc.)…”\n3.3 Conversion of proconvertase to convertase is mediated (I think - not clear in\nmethods) by incubation of proconvertase with \"preactivated C1s\" but evidence of\ncleavage is not provided.\nIn contrast to pro-convertases, convertases are instable bimolecular complexes with a\nhalf-life time of 1-2 min [4,5]. C2 in the presence of activated C1s is cleaved completely\nat room temperature after 30 seconds. Thus, our method was focused on a very short\n(30 seconds) incubation time of C4 and C2 in the presence of activated C1s followed\nby flash-freezing the sample on the cryo-EM grid.\nThe resulting particles in cryo-EM unequivocally indicate cleavage of C4b2 forming\nC2b bound through its MIDAS in the VWA domain to the C-terminal end of C4b, as\nexpected based on the structures of the alternative pathway proconvertase and SCIN-\ninhibited convertase.\nWe added Supplemental figure 6:\nA gel showing complete cleavage of C2 into C2a and C2b by activated C1s within 30\nseconds is added in panel A.\nIn panel B, the chromatogram shows cleavage of C4b2 (green) forming C4b2b and\nfragments C2a, C2b and C4b (black).\n3.4 To generate convertase-substrate complexes, C3-C4b complexes are first\ngenerated by mixing the two proteins together with a cross-linking nanobody. Again,\nwhat is the evidence that this artificially generated complex adopts a conformation\nrelevant to the natural convertase? This complex is then incubated with inactivated\nC2 and preactivated C1s to yield a nanobody-stabilised C4b2bC3 complex. once\nmore, what is the evidence that this complex is representative of the native complex?\nsupplementary figure 6 shows gels of the C3-C4b-nb complex but not of the complex\nincorporating C2b, essential for the characterisation.\nTo circumvent the problem of the short-life time of the convertase, we first pre-formed\nC4b-C3 complexes using the nanobody linker (thereby enhancing the binding affinity\nof C3) and then added C2 and preactivated C1s for 30 seconds and the immediately\nflash-froze the sample (as described in the material and methods). The correct\nformation of the convertase complex is evident from the C2b conformation and\npositioning onto C4b and binding of the scissile loop in a fully productive manner.\n3.5 Minor points:\nFigure legends are brief and would benefit from more explanation for non-experts;\nSome annoying abbreviations - for example, line 68 and elsewhere - \"resp.\" for\nrespectively, should be eliminated.\nWe checked the figure legends. We think that Figure legend 4 describe fully what is\nshown using descriptive phrasing (though indeed brief and without repeating the main\ntext). We agree to add to Figure1-3 and 5 the following:\nWe added in the legends to figures 1-3 , line 725, 733 and 742\n“180°-rotated “\nWe added in the legends to figure 1-3, line 728, 734 and 743\n“from high (blue) to low (red), “\nWe added to the legend of figure 5, line 654 after account:\n“, showing that MG7-surface potentials of C4b-C3 are attractive and C4b-C3b are\nrepulsive.”\nWe have replaced “resp.” by “respectively”."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors addressed the questions raised by this reviewer. It's the authors' choice to use the revised nomenclatures of\nC2a and C2b. On another issue about the protein polymorphisms of C4A and C4B, it was unfortunate that the authors\nignored the phenomenon.", "reviewer_3": "This is an important paper and should be published.\nThe authors have addressed most of the reviewer concerns, although in some cases in a rather perfunctory manner.\nI remain concerned about the justification provided to chose the C4b2b nomenclature; indeed, this was \"proposed\" by four\nindividuals well known in the field but never decided upon by the community. The term C4b2a is historically correct and is\nstill predominant in the literature. It is a pity that this \"proposal\" has further complicated an already difficult nomenclature.\nHowever, this is not the fault of these authors and at lease now they note that this is not generally agreed (in fact should state\nthat).\nThe defence of the stabilised convertases remains rather unconvincing (cross-linking and nanobody) but likely does not\nimpact the data analysed.\nIn my opinion the Figure legends remain too brief and poorly informative - it would have been an easy fix to add a few lines\nto enable legend to properly explain figures."}, "author_response": ""}]

Decision letter