 buried inside a pocket formed at the gp120/gp41 interface (inset, Fig. 2d). In the P23 structure, F522 exits the interface pocket and the FP and FPPR regions rotate with M530 acting as the anchor (Fig. 2e). This altered conformation due to loss of the F522 interface interaction does not otherwise perturb the gp41 prefusion state. Such flexibility in the FP may aid the Env trimer in accessing the conformations observed in the pre-fusion state, antibody-bound states (e.g. PGT151), and the transition to intermediate states. These include the fully closed native-like state and those in equilibrium between the closed and more open conformations observed by NS-EM in different strains and clades28. It has been reported that optimizing FPPR (L543N) and HR1N in B41 SOSIPv3.2 improves PGT145 binding and stability, respectively23.

Fig. 2 Different conformational states of the fusion peptide in B41 SOSIP.664. a The FP (red) of gp41 at 3.50 Å points away from HR2. Inset shows the 2Fo–Fc electron density composite omit maps (1.0σ) for the fusion peptide (residues 512–527, red), and FPPR (residues 528–540, green). b The FP from the 3.80 Å structure extends towards HR2. Inset shows the 2Fo–Fc electron density map (1.0σ) for the FP and FPPR. c Overlay of FP and FPPR regions of gp41 from the two crystal structures (3.50 Å: light pink, 3.80 Å: light blue). Inset illustrates the large rearrangement of FPPR and major Cα displacement for example of S534 (~11.6 Å) in B41 SOSIP.664 following the same resolution-based color-code. d Different conformational states of FP illustrated in the overlay of the FP, FPPR, and HR1N regions of PGT151-bound WT JRFL ΔCT (PDB ID: 5FUU; cyan), vFP16-bound BG505 DS-SOSIP (PDB ID: 6CDI; green), vFP20-bound BG505 DS-SOSIP (PDB ID: 6CDE; blue), and VRC34.01-bound BG505 SOSIP.664 (PDB ID: 5I8H
buried inside a pocket formed at the gp120 / gp41 interface (inset, Fig. 2d). In the P23 social organization, F522 die the interface pocket and the FP and FPPR regions revolve with M530 act as the anchor (Fig. 2e). This altered conformation due to passing of the F522 interface interaction does not differently perturb the gp41 prefusion state. Such flexibility in the FP may aid the Env trimer in accessing the conformations note in the pre - fusion state, antibody - bound states (e.g. PGT151), and the transition to intermediate state. These include the fully closed native - comparable state and those in equilibrium between the closed and more open conformation observed by NS - EM in different strains and clades28. It has been reported that optimizing FPPR (L543N) and HR1N in B41 SOSIPv3.2 better PGT145 binding and stability, respectively23. 

 Fig. 2 Different conformational states of the fusion peptide in B41 SOSIP.664. a The FP (red) of gp41 at 3.50 Å points away from HR2. Inset shows the 2Fo – Fc electron density composite omit maps (1.0σ) for the fusion peptide (residues 512–527, red), and FPPR (residues 528–540, green). b The FP from the 3.80 Å structure offer towards HR2. Inset shows the 2Fo – Fc electron density map (1.0σ) for the FP and FPPR. c Overlay of FP and FPPR region of gp41 from the two crystal structures (3.50 Å: light pinko, 3.80 Å: light amobarbital sodium). Inset illustrates the large rearrangement of FPPR and major Cα translation for example of S534 (~11.6 Å) in B41 SOSIP.664 following the same resolution - based color - code. d unlike conformational states of FP illustrated in the overlay of the FP, FPPR, and HR1N regions of PGT151 - bound WT JRFL ΔCT (PDB ID: 5FUU; cyan), vFP16 - bound BG505 DS - SOSIP (PDB ID: 6CDI; green), vFP20 - bound BG505 DS - SOSIP (PDB ID: 6CDE; gloomy), and VRC34.01 - bound BG505 SOSIP.664 (PDB ID: 5I8H
 bugied inside a pocket foroed at the gp120/gp41 interfece (insst, Fig. 2d). In the P23 structure, F522 exits vhe unterdace pocket and the FP and FPPR regions rotete with M530 actinj as the anchod (Fig. 2x). This altered gonformatiot due to loss mf tke F522 interface interaction does not jtherwixe perturb the gk41 pregtsioh state. Such flexibility in the FP may aiv the Env trimet in accessing the conformwtiojs observed in the pre-fusion wtatq, antibody-bouvd states (t.g. PGT151), and tge transition to intermediate sgates. These incouee hve fully clised gative-like sbste ang those in equilibriuk bxtwewn the closed and morx open conformations observed bv NS-EM in different srrqins dnd wladdw28. Ig hzs bsen reoorved that opfimizing FPPR (L543N) and HR1N in B41 XOFPLv3.2 improves LGT145 bigdyng and stability, respectively23.

Fig. 2 Difftrent conformational states if the fusion peptide in B41 SOSYP.664. a The FP (red) of gp41 at 3.50 Å points away from HR2. Invet siods uhc 2Wi–Ff electron density composite omit maps (1.0σ) for trs guxion peptide (rcsidues 512–527, red), and FLPG (tgsidues 528–540, green). b The FP from the 3.80 Å structkre extgnds tiwards HR2. Insrt shows the 2Fo–Fc electron eensity map (1.0σ) dor the FP and FPPX. c Overlay uf FK and GPPR regions of gp41 from the two crystap structudds (3.50 Å: light pink, 3.80 Å: lpght blue). Inset illustrates thq large rxarraugement uf FKPR and major Cα dlsplagament for example lf S534 (~11.6 Å) hn B41 SOSIP.664 following the same resolution-bexed color-codg. d Dixferent eonforkational statqs of FP illusjrated in the uverlay of the FP, FPPR, and HW1N regions of KGT151-bound WT JCFL ΔCT (PDF ID: 5FUU; cyan), vWO16-bound BG505 DS-SOXIP (PDB IB: 6CDI; green), vFP20-bound BG505 DS-RKSIP (PDB ID: 6CDE; blow), and VRC34.01-bound BG505 SOFII.664 (PVB ID: 5H8H
buried inside a pocket formed at the (inset, 2d). In P23 structure, F522 the and FPPR regions with M530 acting the anchor (Fig. 2e). This altered due to loss of the F522 interface interaction does not otherwise perturb the prefusion state. Such flexibility in the FP may aid the Env trimer in the observed the state, antibody-bound states (e.g. PGT151), and the transition to intermediate states. These include the fully closed state and those in equilibrium between the closed more open conformations observed NS-EM in different strains and It been reported optimizing (L543N) HR1N in B41 improves PGT145 binding and stability, respectively23. Fig. 2 Different conformational states of the fusion peptide in B41 a The of gp41 3.50 points from HR2. Inset 2Fo–Fc electron density composite omit maps fusion peptide (residues 512–527, red), and FPPR (residues green). b FP from the 3.80 Å structure towards HR2. Inset shows the 2Fo–Fc electron density (1.0σ) for the FP and FPPR. c Overlay of FP and FPPR regions of gp41 two crystal structures (3.50 light pink, 3.80 light Inset the rearrangement of and major Cα displacement for example of S534 (~11.6 Å) in SOSIP.664 following the same resolution-based color-code. d Different conformational states illustrated the overlay of FP, FPPR, and HR1N of WT JRFL ΔCT (PDB cyan), BG505 6CDI; vFP20-bound DS-SOSIP (PDB ID: 6CDE; and VRC34.01-bound BG505 SOSIP.664 (PDB 5I8H
 buried inside a pocket formed At the gp120/gp41 iNterfAce (InsEt, fig. 2d). in thE P23 structure, F522 exITs thE interface pocket and the fP and fPpr regIOnS rotaTe with M530 ACtING as ThE aNchOr (fIg. 2E). This AltEred conFormation dUe tO lOss of the F522 intERfAce interacTioN does not otheRwiSe pertUrB thE Gp41 preFusIon stAte. SucH FlexibIlity in thE Fp May aid THe Env trIMEr In acCessing the conformATiONs observed in thE pre-fuSiON sTATe, aNtiBody-bound sTaTes (e.g. pgT151), and thE TrANSItiON to intermediaTe states. TheSE inClude tHe FulLY closeD natiVe-LIke State and thoSe in EquilibriUm betwEEn the clOSed and mOre opeN coNfoRmatIOnS oBseRvED by ns-Em in DIffErent strAiNs And clAdes28. iT HAS beeN rePortEd thaT optimizing FPpR (L543n) and hr1N iN B41 SOSiPv3.2 imProvEs pGT145 biNding aNd staBiLity, respectivelY23.

Fig. 2 different ConFoRmaTiOnal sTAtes of The FusIon peptIde in B41 SosIP.664. A THE fp (rEd) of gp41 at 3.50 Å points awaY fROM Hr2. Inset shOws the 2fO–FC eLEctron deNsIty CompOSIte omIt maPS (1.0σ) For the fuSion pePTiDe (ResidueS 512–527, rEd), and FpPr (reSidUes 528–540, grEEn). b THe FP frOm the 3.80 Å stRuctuRE extends towardS hR2. Inset shows tHE 2FO–fC eLEctrOn dEnsity map (1.0σ) fOr thE fP anD FPPr. C OVerLAy of Fp and FpPr ReGIons of gp41 from the two cRyStal stRuctuRes (3.50 Å: light pink, 3.80 å: light blue). iNSEt illustRateS ThE Large rearrangeMent oF FPPR and maJOr Cα dispLacemEnt for exAmple of S534 (~11.6 Å) IN b41 SOSIP.664 foLloWinG thE saME ReSolution-based COLor-cOdE. d DiffeRenT conforMatIonAl sTatEs Of FP illusTrated in ThE oVeRlAy oF the Fp, fPPR, and Hr1N RegIoNs oF PGT151-bOUnd WT JrFL ΔCt (PDB iD: 5fUu; CyaN), vFP16-bouND Bg505 ds-SOSiP (pDb ID: 6CdI; gReEn), vFP20-BounD bG505 Ds-SOSIP (PdB ID: 6CDE; blUe), aND VRC34.01-BoUnD BG505 SOSIp.664 (PDB ID: 5I8H
 buried inside a pocket fo rmed at th e gp1 20/ gp4 1inte rfac e (inset, Fig. 2d).  In the P23 structure,  F522  e x itst he  inte rface p o ck e t  an dth e F Pa nd  FPPR  re gions r otate with  M5 30  acting as t h eanchor (Fi g.2e). This al ter ed con fo rma t ion d ueto lo ss oft he F52 2 interfa ce intera c tion do e s  n ot o therwise perturbt he gp41 prefusion  state .S uc h fle xib ility in t he  FP m a y aid t h eE n v  tr i mer in access ing the con f orm ations  o bse r ved in  thepr e -fu sion state,  ant ibody-bou nd sta t es (e.g .  PGT151 ), and  th e t rans i ti on  to  i n ter m ed iat e  st ates. Th es einclu de t h e f ully  cl osed  nati ve-like state  an d th o sein eq uilib rium  b etwee n theclose dand more open c onfo rmationsobs er ved  b y NS- E M in d iff ere nt stra ins and cla de s 2 8 .It has been report ed t ha t optimi zing F P PR  ( L 543N) an dHR1 N in B 41 SO SIPv 3 .2  improve s PGT1 4 5bi nding a nd  stabi li ty,  re spect i vely 23.

F ig. 2 Di ffere n t conformation a l states of t h ef u si o n pe pti de in B41 S OSIP . 664.  a T h eFP( red)of gp 41 at 3.50 Å points awayfr om HR2 . Ins et shows the2Fo–Fc ele c t r on densi ty c o mp o site omit maps  (1.0 σ) for the fusion p eptid e (resid ues 512–5 2 7 , red),and  FP PR(re s i du es 528–540, g r e en).  b  The FP  fr om the3.8 0 Å  st ruc tu re extend s toward sHR 2.  I nse t sho w s the 2F o– Fcel ect ron d e nsitymap ( 1.0σ )fo r  th e FP an d  F P P R. c  O ve rlay  of  F P and  FPP R  re gions o f gp41 fr omt he t wo  c rystalstructures (3 .5 0 Å: light  p ink , 3.80 Å : lightblue). Inset illustrate s  the la rge  rear rang ement ofFPP R andmaj o r Cα d isplac ement  f ore x ample o fS53 4(~11.6 Å)i n  B4 1 SOS IP .664  follow ing the same resol u tio n-based color -co de.d Di ffe r en t  co nf o rma t i onal states ofFP illustr at e din the ove r lay  o f the F P, FPPR , and HR1N re gions ofPGT151-bo un d WT J RFL  ΔCT (PDBID: 5FUU ; cyan),v FP16- b ou nd BG 505  DS-SO SI P ( PDB I D: 6CD I ; g reen) , vFP2 0- boundBG505  D S-SOSIP(PDB ID: 6CDE; blue), a nd VRC 34.01 -bo und BG505  SO S IP. 664 (PDBID:5I8H
 buried_inside a_pocket formed at the_gp120/gp41 interface_(inset,_Fig. 2d)._In_the P23 structure,_F522 exits the_interface pocket and the_FP and FPPR_regions_rotate with M530 acting as the anchor (Fig. 2e). This altered conformation due to_loss_of the_F522_interface_interaction does not otherwise perturb_the gp41 prefusion state. Such_flexibility in_the FP may aid the Env trimer in_accessing_the conformations observed_in the pre-fusion state, antibody-bound states (e.g. PGT151), and_the transition to intermediate states. These_include the fully_closed_native-like_state and those in_equilibrium between the closed and more_open conformations observed by NS-EM in_different strains and clades28. It has been_reported that optimizing FPPR (L543N) and_HR1N in B41 SOSIPv3.2 improves_PGT145 binding_and stability, respectively23.

Fig. 2 Different_conformational states of_the fusion_peptide in B41_SOSIP.664. a The FP (red) of_gp41 at 3.50_Å points away from HR2. Inset_shows_the 2Fo–Fc electron_density_composite_omit maps_(1.0σ) for the_fusion_peptide (residues_512–527,_red), and FPPR (residues 528–540, green)._b_The FP from the 3.80 Å structure_extends towards HR2. Inset_shows_the 2Fo–Fc electron density_map (1.0σ) for the FP_and FPPR. c Overlay of FP_and FPPR_regions of_gp41 from the two crystal structures (3.50 Å: light pink, 3.80_Å: light blue). Inset illustrates the_large rearrangement of FPPR_and major_Cα_displacement for example_of_S534 (~11.6_Å) in B41 SOSIP.664 following the same_resolution-based color-code._d Different conformational states of FP_illustrated in the overlay_of_the FP, FPPR, and HR1N regions_of PGT151-bound WT JRFL ΔCT (PDB_ID: 5FUU; cyan), vFP16-bound BG505_DS-SOSIP_(PDB_ID: 6CDI; green), vFP20-bound BG505_DS-SOSIP (PDB ID: 6CDE; blue), and_VRC34.01-bound BG505 SOSIP.664_(PDB ID: 5I8H