Structure of RSV Fusion Glycoprotein Trimer Bound to a Wal ience Prefusion-Specific Neutralizing Antibody Jason S. McLellan et al. Science 340, 1113 (2013); DOK: 10.1126/science.1234914 This copy Is for your personal, non-commercial use only. Ifyou wish o distribute this article to others, you can order high-quality copies for your colleagues, clients, or customers by clicking here Permission to republish or repurpose articles or por following the guidelines here, ns of articles can be obtained by The following resources related to this article are available online at wwnw.sciencemag.org (this information is current as of January 9, 2014 ): Updated information and services, including high-resolution figures, can be found in the online version of this article at: http:/wew.sciencemag.org/content/340/6136/1113 full html Supporting Online Material can be found a: http:/hwwew sciencemag.org/content/suppl/2013/04/24/science. 1234914.DC1 htm! This article cites 49 articles, 20 of which can be accessed free: http:/Avww.sclencemag.orgicontent/340/6136/1113.ful.htmiref-list-1 This article has been cited by 10 articles hosted by HighWire Press; see: http: Awww. sclencemag.org/content/340/6136/1113 full Mmiffrelated-urls, This article appears in the following subject collections: Biochemistry http: thw. sciencemag.orgicgi/collection/biochem Science (print ISSN 0036-8075, onlin ISSN 1095-0203) s published weelly, except the last week in December, by the ‘American Association for the Advancement of Science, 121 2013 by the American Association for the Advancement of Science; all rights reserved. The lille Science is a Tegistered trademark of AAAS. New York Avenue NW, Washington, DC 20005. Copyright org on January 9, 2014 sciencemag. Downloaded from www.TT i a IS inca posacee ICENP-Gynucleosome] Fig. 4. Hydrophobicity of the CENP-A tail is the primary determinant for of the CENP-A nucleosome by CENP-C. Isothermal t- tration calorimetric curves are shown for binding ofthe human CENP-C cenbal region (CENP-Cyay2s7) and the CENP-C motif (CENPG_ 3) to mcecsores containing #3;-a3rA8EGLG (human) and H3y-332- OF (budding yeast), respectively CENP-Cyy 4s fragment, which contains the CENP-C motif and the dimerization domain, is capable of bing targeted to the centromere when ‘overexpressed (20), In Xenopus, chromatin in ‘which IADB is replaced by protamines docs not support CENP-C localztion to the om tuomere of sperm (9, 27). However, CENP-C colocalizes with CENP-A upon addition of ex cect, because the histone chaperone nucle plasmia catalyzes the eviction of protamines and the incorporation of H2A/HDB dimes into the sperm chromatin (22). The funetion of hu rman CENP-A eam be complemented by the bud ding yeast Saccharampoes cerevisiae hammlog, sot 22) (Fig. 4), Our study abo sugges that CCENP-C can recognize CENP-A chromatin, through malivalet interactions, allowing itt associate more strongly and selectively (figs S14 and S15) (20, 23). In adton, our resus have implications for various strvtural models of the centwmerie nucleosome, a widely dis cussed and controversial topic (24), The finding thatthe broadly conserve CENP-C most in a dimeric CEND-C could bind to two IDAAL2B. tnd two CENP-AM4 molecules constrains these models because CENP-C is consitvely asso- ciated with the centromeric nucleosome. Our study provides the strctural basis forthe re anion of the CENP-A octameric micleosome by CENP-C. Further understanding of how it ‘engages chromatin should provide important Insight into vored compositions of CENP-A, ‘nucleosomes. (Owe finding thatthe widely conscrved CENP-C no binds to te eentemenc mackerel ‘with the rest identification ofthe budding yeast homologs ofthe vertebrate centromere proteins CENP-T and CENPAW (25, 26), suppor con served mechanism of centromere targeting by the kinetochore, Furhemore, our study has ro vealed a histone recognition mode whereby an intrinsically disordered peptide binds to the his tone til through hydrophobic interactions foil. itted by nucleosome docking; this broadens the repertoire of the cll to “read” histone variations (07,28), 0 ony a the centromere but poss ‘also in other contexts, References and Notes 2. Stet AC Rad, HS. Wal, Sac 293,196 Goon, 21.5. Verasdn, Kia, Hate CB 2 320 bon. ‘hom et, C335, 039 oD. WR Potts Cur Bi. 2.399 2031) EW Cat | i Shan Cal Bo 29, 143 Gora 6 Ae, CW. Caa Mae C1 Fler, KF Stig otve 477,94 oO. 2. E Seep et a Car. i 23, 391 On. Son, 8 Ganon, Wa agi, Toe, 9A, ee, A. tah Mo iC 20, 10. K Tana HL chang A. Kagan ata, Dee ca 7, 334 2009, 21, Sec ef a, Gees Da 19, 2081 2005. ZL Gah ay el Bal C9, 40 (00, 13, CW. Gal MCS KML GLE rn, AF sual et Cl Bol 31.695 03. 1 Vegan, Vans, Kay Pte 3,08 ‘e006, 15, Wate, Pe Mal ced Se US 8 08, 12283 om, 16, We Tabane a Mate 476, 232 00 7A Bate of See 31, 856 (2000, 0 BO. Mae 1Glan, HP ema, 5.1, 29. fc] mad 1D Care . Cz) Na, TCE fongsen See 334 977 CO REPO 20. 5 ama a St, 99,9 20 2 ip tom Co 6,158 0992). 126 a Oty, Obert 5 Deka, P terme Ce Be 24,8620 200. 2, Std Yoga Oka, Ao, It eo 2,229 a) 24, BF fl WG el 244 A710 2. CF Baker, Aa. Ca Bel 4, 619 O12, BE Sere aly Eo Ba 404 O12, 21 taste, Car Opn Se ot 20.794 Gow, 2a Cok Mase Eade, 16 Hae, Atenladgens: We nk sl the Manan Sa 90310 smi er eri par Cr Ftcpatn fhe pope | bir maser es ‘Rly Cty and He ones fra an taro han ere lari rd ae for CA Feclanane retin or 60 OU Scopes by the ere roan othe Rata ce tia A, HF 2 ard V8 he hat te ‘Alay ares Does att 1520, Be Rata Ingo etter gee ary Us gn 7 GTE726 OUR we KES an ara act Ise got YEO We sae Gr Sines govt TG 104 nd Us Orpen of ery pn Ae 06113571. Te Se FID 6 AN Supplementary Materials smmcancenon want 4060360310002 Tha sttess fete 29-5) 23 ow 2015; acai ol 2003 SaPerione 23552 Structure of RSV Fusion Glycoprotein Trimer Bound to a Prefusion-Specific Neutralizing Antibody Jason 5. McLellan,"* Man Chen," Sherman Leung,” Kevin W. Graepel,? Xiulian Du,* ‘Yongping Yang,* Tongqing Zhou, Ulrich Baxa,* Etsuko Yasuda,? Tim Beaumont,” ‘Azad Kumar, Kayvon Modjarrad,? Zizheng Zheng,* Min Zhao,* Ningshao Xia,* Peter D. Kwong,** Barney S. Graham* ‘he preusion state of respiatory syncytial vius (RSV) fusion () glycoprotein isthe target of most RSV-neutralizing activity in human sera, but its metastability ha hindered characterization. To overcome this obstacle, we identified prefusion specific antibodies that were substantially more potent than the prophylactic antibody patvizumab. The cocrystal structure for one ofthese antibodies, 025, in complex withthe F glycoprotein revealed 025 to lock Fin its prefusion state by binding to a quaternary epitope atthe trimer apex. Electron microscopy showed that two ‘other antibodies, ANZ22 and 5C4, also bound to the newly identified site of wulnerbility, which we named antigenic site @. These studies should enable design of improved vaccine antigens and define new targets for passive prevention of RSV-induced disease cspimtory syncytial virus (RSV) infess nearly all children by 3 years of age (I) and i eating cause of infant hospital ization and childhood wheezing (2, 3), Globally, RSV accounis for 6.7% of deaths among infinis 1 month to year old more than any othe sin tele pathogen except malaria (4). The only inter ‘vention is passive administration ofthe licensed ‘monoclonal antibody palivizumab (Synagis), ‘which recognizes the RSV fision (P) gheoprotcin (6,0) and reuces incidence of severe disease (7). Clinical evidence that RSV F-specificanibodis can protet pains! disease has prompted a search {or beter antibodies (8-10) 2nd a concerted elon to develop an effective vaeciae (1). RSV F is type I fusion protein (12) dat rearranges fom metasiable prefision confor: mation to highly stable postfusion strcture. wuwaciencemag.org SCIENCE VOL 340 3° VAY 2013, RTS i 113EPORTS Three previously described antigenic sits (LI san V) associ with nealing atv (13-15) exist on the pestfason form of F (16, 17). Ab- soxption of man sera with pstfision F however, fails to remove most ofthe F-spocifienetrelzing activity, suggesting that thee are neutralizing sntgenc sites uaigue tothe prefision form (18). ‘Thus, detemnining the prefision RSV F strocure and idenilying antibodies that bind prefasion- spastic antigenic sites have become conversing ores for developing new anions and ac ins to prevent RSV infection. From mice immunized with gene based veo- tors expressing the proein (19), we isolated an antibody, SC4, that was 50 times 35 potent 35 palivizuma (Fig. 1A) and did no bind to sol ble form of RSV F stabilized in the postion confirmation (76) (Fig. 1B). We determined that 'SC4 shared these properties with two reveal ly isolated human antibodies, D25 and AM22 (10, 20,21) (Fig 1, and B), and we hypoth sized that these antibodies recognized the meta ‘table prefusion conformation (22) ‘We focused our structural efforts on the hu- ‘man antibodies by frst sereening their binding 0 pane of RSV F glycoprotein variants (23). We observed D25 and AM22 antibody binding 9 a acne Rear Cn, Natori of Alergy ard ela Dou Attlee ea ahd 20092 USA "contra aber Adan eo telgy Peron SI Fedrc In, Fede son ab rater Cnc sere eon MO 2702 USA “AMM Theapetis, ade Medial Cris, Amsterdam, Neher lors sna tate ef Dts ad Vac Dop- rertn bis Dba, men Urry, Kaen, Cina, e105. ‘onezpondig author. Eat melanin@riaidcihgon Sin; pawonoe@nit or DK) Fig. 1. RSV neutralization, F glycoprotein rec: grition, and rystalstrctre of human antibody 'D25 in complex with the prefusion RSV F trimer. ‘The pretusion conformation of RSV Fis metastable, and wen expressed ina souble for ready adopts the postion state; a numberof patent antibodies, induding 025, bind to a newy revened antigenic Site at the top ofthe preusion Fgycopatein. SV neutralization by antibodies. Palvumab & the US. Food and Brug Adinstation (FDA)-approved ‘roplnctc antibody that prevents severe RV dis ‘xe. (B) Emyme-inked immunosorbent ay mea suring antibody binding to postion Faycoproein, For (Nand (8), data are representative of multiple independent experiments. (© D25-RSVF timer cys tal srucur in ibbon and molecular suiace repre Sesion. One proto ofthe Fghycoprctein timer show as bons and colored 2 2 tainbow rom tie to red, W terminus of Fy to teri of Fy, respective. Molecular srfaces are shown for the other two Fprotomes, colored pink and geen The 'D25 Fab bound tothe F protomer shown in ribbons is aso played in sbbon representation, with the hay cian colored red and tight chain colored gray. The ether D25 Fab are colored the sae, but shown in srface representation, 114 construct, RSV F(+) Fd comprising residues 140 513 fised to a C-terminal flim trimerntion domain (24) However, 8 filed to form com plexes by mixing purified RSV (+) FA with purified 25 or AM22, suggesting thet F was {nigger during purification (23) T capture Fin its prelasion state, RSV F(*) Fd was expressed as complex with D25. Optimal expression was ‘oblained rom cotrnsfection of DNA encoding 1D2S Fab with DNA encoding RSV F(+) Fd (lig St), X-ray diffacton data to 36 A resolution ‘were obaaned on crystal ofthis complex, and the siiucure was solved by molecular replacement tising the unbound D25 Fab since (able St) and portions of the postision RSV F simcture (06, 12) search models. The sructre was te- fined 10 Reya/Rewe of 21.326:7% (Fig, 1C and table St). ‘The D2S-bound RSV F structure resembled the prefision stucre ofthe rele purinllvenza vis 5 (PIV) F glycoprotein (26,27), indicating that D25 binding stabilizes RSV P in the pre fusion conformation (fg. $2}. Comparison with the postiusion RSV F glycoprotein structure (6, 17) revealed that most ofthe secondary and trary structure was preserved in both pre-and post fasion states, with 215 residues showing less than 2A Ca. deviation between the two sirvtures (Fi, 2) In contra, egos at the Nand C tein ‘ofthe Fy subunit showed marked conformation: al changes. The fusion peptide, located atthe [N terminus of Fy, and five secondary structure elements (22, 03, 04, and dhe 3/4 hip) re ‘arange and fse withthe a het to frm sin- ‘le extended postfision helix (25a) of 100 A in length fig. $3). At the C terminus of F,, the sole parallel srand (622) unravels, allowing the prefsion 10 helix to move toward the OS pu. A SV nralaton nee Palwaumab | ‘o0\nin vom bln. Similar rearrangements are observed in the comparison of prefsion PIVS snd. postision parainfiuenza vis 3 (PIV3)F plycopotein sruc- tures (27), indicating that F glycoproteins fom both the Paramyzovirinae PIVSPIVS) an Pew ‘movirinae (RSV) subamies undp sila om ‘oemational earangementsofaciiate membrane fision. Despite overall similarities between the PIVS sand RSV F profsion structures, thee are distinct ferences important for function and antigen- icity. In the refasion RSV F structure, the els- looprhelix (a6 and a7) moti that constitutes antigenic ste Ils located frther away from the three-fold imer axis than isthe homologous region in PIVS F (lig. $4). This repositioning exposes the face of the helix Toop-hels hour by palivizamab and motavizumab, allowing these antibodies to bind prefusion RSV F without fst, uring conoratonal reerangeneat Wo occur originally suggested on the basis of modeling ‘with the PIV F stucstre (28) ‘Anouher difference is thal the RSV F fasion peptide is brie in the contr of theteimer cavity, ‘with ts N terminas located more than 40 A aay from the lat visible F> reside (Fig 2). In eon- tat, the PIVS F fasion peptide lis in a surice groove betwoon subunits, is patlly exposed 10 solvent (27) and undergoes minima movement after protease cleavage (26), Ths suggests that in RSV F.a subsantl souctural earangement of the fusion peptide occurs after the Fy pre- cursor is cleaved by the frin-lke host protease to produce Fy and Fy subunits (29, In compar: json to other sructures of cleaved, typeI fusion proteins (26, 32, 37), the locaton ofthe RSV F fsion peptide is most similar to that of influ Fbemagaluinin (3) (fg, SS), whichis surprising eee ae Favs sve fener 31 MAY 2013 VOL 240 SCIENCE www.sciencemag.orgven that hemagglutinin is triggered by acidic DH inthe endesome, wheres RSV Ftrigerings plindcpondent and thought to occur atthe cell sure (32) Inthe RSV F prefuson timer, the C-terminal hice (010) for an inverted pyramid (lig. Sa) Ta contrast, the C terminal helices ia the PIVS F trimer forma coiled col, which was stabilized by coiled oil GCN4-timerization mf 27) The ‘addon ofthis mos was sufficient to stabilize PIVS and human metgpneumovines (HMPV) F protins inthe prefusion state (27, 33) The verted pyramid conformation observed in the RSV F sactre may explain why RSV F could note expressed with the GCN4 mf but could bbe expressed with the Mbit trimerzation d= main (24, The action ofthe ibrtin domain was not, however, sulci to suiize RSV Fin the prefision state, suggesting that it is not an optimal substitute forthe native transmembrane domains that normaly stabilize in the vir brane (34. The binding of antibody D2S was ths requiedt stablize the proision timer 'D2S recognizes a quslernary epitope atthe rmembrane-dstal apex ofthe RSV F glycopmatcin (ig. 1C). The D25 heavy chain interacts with fone prosomer (involving 638 A? of interactive surf ateaon F, and the D2S light chin binds to thosame prowomer (373 A?) and a neighboring Rev Fira| Movoment>5 A Pretuon F prot (112A? ig. 34), D2S cons RSV Fh eof iss open orang pons (CDRS) whe hay nd CDR Imac wih he hel esc 1960 210)and forming inemolsurhyroge bonis ith nds 63, 65, 66 and 68 in the oop ‘vce snd Band Hell, Wheres be secon soc of the D2S elope remais trol ung a pre-and pois con teations, he eriny suc changes ob tantly, thetic pivoting 180" rave tostand 2 (i, 3B), Thisexpan the flare {1 D25 to bind postion ¥ ad suggest that 125 neal RSV by ing Fs pref confemaca, 25 bins he eas conserved ego onthe F glycoprotein (Fig. 3C). Although F proteins from human RSV A and B sates ih ated Fn soqcve (47 of 472 47% fe sno Se coming the nature Fy, ean drei) tly ee ae vai tons ae looted in rn bun by DOS SD) Shar tthe 50 ri weir ove RSV F thr ot Kate be sie min offuman RSV Fsbo A Sa fn tise roan e.3D) Th te DPS epg, Sti ope of he pion RSV Fst ay te unt immune peso a save a4 dt mina pespoiimuniy (5, 30) REPOR To investigate whether AM22 and SCS bind the D2S eppe, we petormedl compton binding and electron microscopy (FM) experiments, We ‘éctemined that DDS binding to RSVnfected ells was competed by AMZ2 axl 5O4 (Fig. 44). In ation, pegativestain EM images of Fab D25- SV F complexes resembled the EM images of Fab AMZ2 RSV F and Fab SCA-RSV F complexes (Fig. 48). Together, ese results sugges that an Aibodies D25, AM22, and SC4 recognize the same or highly related epitope, which we named “antigenic site ©.” In ation to their extraed- rary potency and prefision specificity (Fig. 1A), all he antibodies strongly ibe fasion when add after atachment (Fig, 4C), and all three ‘were unable to block cll surfice attachment (Fig. 4D), suggesting thatthe pustive RSV F receptor bins to a region on F not block by hese a= bodiss, AMZ2 and D25 antibodies also neutral ized similarly in both Fab and immunoglobulin contents (Bg, 86), inicating thal avidity was not ‘aquired for potent neutlization, in cont 10 ‘what was previously observed fr some influenza vin anbodis (37, Thus ntgeie se © spite antibodies are distinet from neutraliing anti- bodies to ther known antigenic sites on RSV F Docause of their exclusive recognition of the prefsion F structure and extemely high potency (ie 87). Fem com 1p, F, Cem Prefuson F Postion F Postion protomer proton ‘emer Fig. 2. Structural rearrangement of RSV F. To mediate vrus-cl entry, the RSV F glycoprotein transitions from a metastable presi conformation to a stable postuson conformation. Outer images display prefusion (eft and postfusion (right) trimeric suructres, colored the same as in Fig. 1C. A ‘complex glycan, shown 2s sticks, is modeled at each ofthe three Mined ‘costo sites found inthe mature protein. Inner images display a single SV F protomer in rbon representation, colored asa rainbow fam blue to red, N terminus of Fy to C terminus of Fy, respectively. Select secondary. structure elements are abled (correspondence with amino ac sequence in ie- and postion conformations is shown in fig 2) Inst: Schematic ot the mature RSV F protein in the RSV Fs) Fl consct. The rainbow coloring of the boxes representing the Fp and F subutts matches tht In the Structures, Gycans ae shown as branches on top ofthe boxes, and dstide tends are shown as black Hines under the boxes. Amino acs that move mae than 5 Ain the pre-and postion contarmations are indicated by lack bas wuwasciencemag.org SCIENCE VOL 340 2° VAY 2013, RTS i 115P 1116 ‘ORTS Antigenic site @, loeated at the apex of the profusion F trimer, should be ediy accessible {even oa the crowded virion surface, which may Fig. 3. RSV Finterface with D25. Antibody 025 binds @ quaternary epitope spanning two roto: mers at the apex of the prefusion F trimer. (A) Close-up of the interface between D25 and RSV F Side chains fF residues interacting with D25 are labeled and shown as sticks. Oxygen atoms are Colored red and nitrogen atoms are colored blue Hydrogen bonds are depicted as dotted fines. The two images are related by 2 90° rotation about the vertical axis. (8) Position and conformation of the 025 epitope onthe prefusion and postusion F imolecules, RSV F residues at the D25 interface ae colored rec parity fe and as inated with arn, with fragment N and teint ind ated. (© Surface representation of RSV F colored according to sequence conservation. Coloring was generated with the ConSurf server (40) using 1178 F glycoprotein sequences from human RSV sub type A and B, as wel as bovine RV. (D) Sequence conservation of F resides in regions recognized by 125. Amin acs in human RSV subype B CHRSWB) ‘in bovine ASV (RSV that fer for RRSWIA are ‘aloe te. Ectaomain defined as resis 26 to 109 and 137 to 524. Abbreviation fr the amino acid resiues are a follows: A, Al; C Cys , Asp: E (lu; 6, Gly H, Hs I eK, ys Leu N, A , Pro; ln, Are Sex, Th and, Val Fig. 4. Antigenic site ©, Highly eflecive RSV rneualiing antibodies target site ate membrane: Atal apex o the prefsionF times. (The ability of antibodies to blck 025 binding to RVnfected ols vas measured asa function of antbady concentration (@) nats of RSV fab complexes by negatvestain deton mkrocopy (EN: Reptoecton ofa 32 A slice through the arta structure of RSV F-+ 025 Fab filtered to 10 & resolution le slice was use to emphasize visibility of the F glycoprotein cv Aligned average of 263 particles of RSV F + D25 Fa (nile lf. Aligned average of 50 particles of SV + AMZ2 Fab (ile igh. Nignd average of {171 particles of RSV F + 5C4 Fab (ight) Scale ba, 50 A. (Q Fusion inhibition and @) attachment inhi bition activity for antibodies targeting atigenicsite and Fspoctic antibodies targeting other antigenic sites. For the attachment inhibition asa, heparin was ‘ed a a postive contro. Data in (A, (O, and () are ‘representative of multiple independent experimen. ‘explain the observation that most neutralizing RSV F (18), That the dee antibodies described ‘ctvty in human sera indaced by natural RSV in this sx’ recognize antigenic site © at dif infections direct agains the prfision form of ferent angles (Fi. 4) and share lo soquence A sntwensanennnors — B DA rection Noga sin EM 3 wo = a re gx 4 . Di fr b vy ig g ie voi c Fuse eraion D peace 2 bal ee g Qhigers a 5 py PSUS | levcrat i E ae Param : a” © Repan 19m. ota 31 MAY 2013 VOL 240 SCIENCE www.sciencemag.orghomology suggests that there may be many other antibodies within the repertoire eapable of recog ning this prominent epitope. The high potency of antibodies agsinst antigenic site O suggests that they oul developed fir passive propyl of RSVindced disease in neonates. Notably, the Jimmunogar elicited SC4 aasbody provides proo!: of principle that antibodies against antigenic site © can be induced by gene-based immunization presenting the mative form of F Vaccine antigens Stabilized inthe prefision conformation, perhaps facilitated by linking mobile and immobile por- tions of F with disulfide bonds, may futher im- prove elicitation of antibodies to antigenic site O. Definition ofthe D2S.RSV F stracture thas pro vides the basis For new approaches to prevent RSV.inducod disease, References and Notes eo CH Ta, Fa A Am ‘echt a0, 53 60, Doe Shao a, A 242,240 (950, let Egle, 360,54 (200. tan taf ane 380, 2095 C2) 5. ono nee 276 1215 (997) LA Bee Kan Wye Col Vit 6, 2042 ase. 1. the Me RY Sly Gg, eis 32, 532 590 8 ED Cla tf nme. 13, 638 2009 8M weet aly tm 36865207. 10M | stb at tel 26, 179 201. 11S Geta nmol fe 259, 119 O20 12 EE woth 1 Mask) Wo 4, 172 C983, 13 LMbi tal Gen Wl 73, 2225 90) 14 LA tere lJ iol 72, 622 0998, 45. 1 Lope, B Gaia ane, A Mer, 2 feta Wed 4, 927 1990. 1615. Wellin, Yang 85. aha. Kg, 2 oot ws, H78 GO 17 KA Swann el, Prec Net Aca Sl USA 208, sas oon. 8 MMe a, Pc No Ac Sc SA 309, 3089 on ‘Sie Onin. % a, Sits. Spe ng css 2d man radi he. Patent pean 121600950 2610. Osumont A.B, Yas, RV specie ‘nda mk. Pat apoation 1298.23 012 eprom 1005 greater psy HZ? an 1S eave palvamab sae by Became 2d alleges pao aso er fom based ‘elation any, eased 21 ed igre! ‘ahi ads 3d igor oe Srna Brace, as OME 35702 BOL. Sank eat, oto 308, 081 (0 5S. Chamspegito, Fan, PL Calin, FEM pond WE Pcl LV 85,3966 GOLD. 2.0. Wack, re Aa fed Se USA 308, 1esr2 e012, Sin X. Wen, RG Paterson RA. amb, 15 ey, are 9,30 (208. 15 Mn tf, Ho See Mol Bil. 7, 248 ‘oid, FSV hs teste son peptide ol ‘ramps wich may allo 9 mo fam {nts pre gti othe al ayo ee Te te erat, Aare 454,177 08. 1 Wo, Sal 0. Wey, Nore The fn pepe comeced the are posed {2 and fee toh ona peng cen the potas tat oy 20 Ai dare te opening maybe se 35 xt ah fr een ote deg tigen. JEW tl Mt Soc Mol ua, 19, 63 OL "rating ft Fy Conn code ee ‘her poh tease he ren nea este 46 ‘ona mith he sin pee a neha some Durr wasn he poston ae, Fy ‘etre 16) 0513 ow at ot 8, wih cul bia te len pepe a ad gun F-Canbes Cm ingle A sen J Gen i 73 nu a992). (On the ba of euece aa 2 op gan drs wos pated wot hn he amps ly tat gh be ander ne ese I has been densated tat ino FV sobgrupspcc mcr anos canbe Mec ys etd ans beeen Fes 20 and 216 0) ae that a pepe comeing Fs, ‘Sh, abou 2 pei eape as at dtned 9. REPORTS: | 1, 0. fle tl, nar 49,52 2012. SEAL Goma. Bet, Toms, et Vl 63, 16 goon, 39, Coney Boge abi, Ah We. 142, ton G97 40, 1 ten, fe, Mas Pp, HB a Mae ch es 38 (We Serve S29 (2030, Acknoledgmets: We tak members he Stk Bley ec, Suu Beats Ce Sry a rl Paton abr the Yc Ret eer {er ep meses ood | Ch | cy. Ce, nd sa aS oes Regal aba Ress a) bl wth hacen dat caece he l ested abi manscip are alae ne ae ap! Sed sloery mee ic ees od Se {ean af be mote she bs depts erent ark nde cues os AA OM. Sloper ths wat wos pray be Fara Ro Faye anal tie ef Aegon Ose) ond {Wet atl hc Fn he 16812081 Ue eset dee 22 (SECA ate aed Pot ‘Soe ws spre y he US. Daren of ey as neg Ste, Oe af Swe, es cata 1953130 ng 1 amet an erator pet inten dt cay a rat 23 ‘2 en and edo tng esl bd predic WO200706 "OA EY. aT [sesamiae opti ony 125 spec: gees a6 mans pots they, WORD 9640 an nay 2 SY gee ‘Gna mokel, WOZDLIOSH3A) 153 PDK end BS. oe iets ona US part apcatn desig te teal paetattind RY pop nate ‘an eon AF pets ee 61795389, ISM, Mc. 72. M2, Wan BS se ero ‘hep apt ortny SCae eop ‘ets AS gh ttzon ated nd th pie can (el pet a FSV en adele ease, oie Supplementary Materials somacencra ergleenenatance 234914001 Walang Weds Figs siw 7 Ferre (4-58) aman 200%: aed 25 Har 2073 Fae ote 25 Bp 201 saaPeicene 34914 wuwasciencemag.org SCIENCE VOL 340 2° VAY 2013, W17