doi:10.1016/j.jmb.2007.02.051 J. Mol. Biol. (2007) 368, 791–799

Chloroplasts Assemble the Major Subunit FaeGof Escherichia coli F4 (K88) Fimbriae toStrand-swapped Dimers

Inge Van Molle1,2†, Jussi J. Joensuu2,3†, Lieven Buts1,2

Santosh Panjikar4, Mirkka Kotiaho3, Julie Bouckaert1,2

Lode Wyns1,2, Viola Niklander-Teeri3 and Henri De Greve1,2⁎

1Department of Molecular andCellular Interactions, FlandersInstitute for Biotechnology(VIB), Brussels, Belgium2Ultrastructure Laboratory,Vrije Universiteit Brussel,Pleinlaan 2, 1050 Brussels,Belgium3Department of Applied Biology,P.O. Box 27, FIN-00014University of Helsinki, Finland4EMBL Hamburg c/o DESY,Notkestraße 85, 22603Hamburg, Germany

† I.V.M. and J.J.J. made equal contrAbbreviation used: Nte, N-terminE-mail address of the correspondi

hdegreve@vub.ac.be

0022-2836/$ - see front matter © 2007 E

F4 fimbriae encoded by the fae operon are the major colonization factorsassociated with porcine neonatal and postweaning diarrhoea caused byenterotoxigenic Escherichia coli (ETEC). Via the chaperone/usher pathway,the F4 fimbriae are assembled as long polymers of the major subunit FaeG,which also possesses the adhesive properties of the fimbriae. Intrinsically,the incomplete fold of fimbrial subunits renders them unstable andsusceptible to aggregation and/or proteolytic degradation in the absenceof a specific periplasmic chaperone. In order to test the possibility ofproducing FaeG in plants, FaeG expression was studied in transgenictobacco plants. FaeG was directed to different subcellular compartments byspecific targeting signals. Targeting of FaeG to the chloroplast results inmuch higher yields than FaeG targeting to the endoplasmic reticulum or theapoplast. Two chloroplast-targeted FaeG variants were purified fromtobacco plants and crystallized. The crystal structures show that chloroplastscircumvent the absence of the fimbrial assembly machinery by assemblingFaeG into strand-swapped dimers. Furthermore, the structures reveal howFaeG combines the structural requirements of a major fimbrial subunitwith its adhesive role by grafting an additional domain on its Ig-like core.

© 2007 Elsevier Ltd. All rights reserved.

Keywords: F4 fimbriae; chloroplast-targeting; chaperone/usher pathway;strand-swapping; enterotoxigenic Escherichia coli

*Corresponding author

Introduction

Binding of bacterial pathogens to host cells istypically mediated by adhesins. These are located onthe bacterial surface in polymeric, proteinaceousappendages called fimbriae or pili, or in non-pilusstructures.1 The most prevalent assembly pathwayfor these adhesive structures is the chaperone/usherpathway.2 Regardless of their ultrastructure, allknown adhesive structures dependent on the cha-perone/usher pathway are assembled via a donorstrand complementation/exchange mechanism.3–5

Fimbrial subunits share an immunoglobulin-likefold that lacks the last β-strand, thus creating a

ibutions to this work.al extension.ng author:

lsevier Ltd. All rights reserve

deep hydrophobic groove on their surface. In theabsence of a specific chaperone, the subunitsaggregate in the periplasm and are degraded bythe DegP protease. In a reaction called donor strandcomplementation, the chaperone donates a β-strandto the subunit fold. By capping its hydrophobicgroove, the chaperone stabilises the subunit, helpssubunit folding and prevents aggregation. At theouter membrane usher, the strand of the chaperoneis displaced by the N-terminal extension (Nte) of thenext incoming subunit in the growing pilus.3,6–8

F4 fimbriae encoded by the fae operon are themajor colonization factors associated with porcineneonatal and post-weaning diarrhoea caused byenterotoxigenic Escherichia coli (ETEC). Because theiradhesive property is not located only at the tip, butall along the pilus structure, F4 fimbriae are differentfrom the well-studied, tip-associated fimbriae (e.g.type 1 and P-pili).9 FaeG is the major structuralsubunit and the adhesin of the F4 fimbriae. Al-

d.

792 Crystal Structure of the F4 Fimbrial Subunit FaeG

though recent publications have elucidated manydetails of the mechanism involved in the assemblyof pilus and non-pilus adhesive structures,6–8,10–12the atypical character of the F4 fimbrial systemstill raises questions. Besides combining thestructural and adhesive fimbrial properties, FaeGis much larger than other fimbrial subunits(27 kDa versus approximately 17 kDa) and lacksthe conserved disulfide bridge present in otherfimbrial subunits.13,14

There are three naturally occurring serologicalvariants of F4 fimbriae: F4ab, F4ac and F4ad.15 Eachvariant exhibits a distinctive binding profile toporcine enterocytes. The amino acid sequence ofFaeG in the F4 variants shares a conserved epitopedesignated a and each variant has a specific b, c or depitope.16 ETEC strain 5/95 is a naturally occurringF4ac strain in which the F4 fimbriae are less stablethan in other F4ac strains, including the referencestrain GIS26.17

ETEC-induced porcine diarrhoea leading toreduced weight gain and mortality is a majorproblem in the pig industry worldwide. To date,there is no commercial vaccine to protect weanedpiglets against F4 ETEC infections. However, oraladministration of purified F4 fimbriae to piglets,induces an F4-specific mucosal immune responseand protects them against ETEC challenge.18 To testwhether an edible subunit vaccine against porcine

Figure 1. DNA, RNA and protein analysis of tobacco plantgene cassettes in order to target the FaeG protein to the apotransformation vector T-DNA regions for apoplast and ER (leftthe hexapeptide SEKDEL was added to the C terminus of theindicating the copy number of the transgene faeG. Ten microghybridized with the faeG PCR fragment. (c) RNA hybridizatiA 9 μg sample of total RNA extracted from the leaves was loaproduced in the transgenic plants. A total of 20 μg of totalcauliflower mosaic virus 35S promoter; SS, apoplast-targechloroplast-targeting transit peptide from pea rubisco smallsynthase gene from A. tumefaciens C58. Numbering refers toconstruct. C, non-transgenic control plant.

F4 ETEC infections is feasible, the expression ofFaeG was studied in transgenic plants.19–21Here, we compare the production of soluble

FaeG in different cell compartments of tobaccoplants and describe a unique dimeric structure ofthe chloroplast-derived FaeG. Knowledge of thestructure of the FaeG subunit allows us to gaininsight in the F4 fimbrial assembly and brings newinformation on the F4 receptor-binding site.

Results and Discussion

Expression of FaeG in different tobacco plantcell compartments

To target the FaeG to the chloroplast or theapoplast, the faeG sequences, coding for the matureFaeG proteins, were N-terminally fused to thechloroplast transit peptide sequence (TP, FaeG5/95and FaeGGIS26) of the pea rubisco small subunit orthe apoplast-targeting signal sequence (SS, onlyFaeG5/95) from the barley trypsin inhibitor. To retainFaeG in the ER, the hexapeptide SEKDEL wasadded to the C terminus of apoplast-targeted FaeGconstruct (Figure 1). The TP-faeG gene fusion re-sulted in additional amino acids at the N terminus ofchloroplast-targeted FaeG. After the cleavage of theTP, four extra amino acids (MDRS) remained at the

leaves (generation T0) transformed with the different faeGplast, ER, or chloroplast. (a) A presentation of the plant) and chloroplast targeting (right). To be retained in the ER,apoplast-targeted FaeG. (b) DNA hybridization analysisrams of HindIII-digested DNA was loaded per lane andon analysis showing the amount of faeG-specific mRNA.ded per lane. (d) Immunoblot analysis of the FaeG proteinsoluble protein from leaves was loaded per lane. p35S,ting signal peptide from barley trypsin inhibitor; TP,subunit; nos 3′, 3′ untranslated region of the nopalineindividual transgenic plants obtained within each gene

Figure 2. Purification of chloroplast-targeted FaeG. (a)Detection of total proteins on SDS-PAGE with silverstaining. (b) FaeG immunoblot of the same samples. timezero, 1 min, 3 min, 5 min and 10 min time-points inminutes at pepsin digestion; 5′s, soluble protein fraction ofthe 5′sample; FaeG5/95, final purification product; F45/95,purified fimbriae from strain 5/95.

793Crystal Structure of the F4 Fimbrial Subunit FaeG

N terminus of chloroplast-targeted FaeG. In the ER-targeted and the apoplast-targeted FaeG one extraamino acid, G or S, was added, respectively.Only transgenic plants transformed with the TP-

FaeG construct yielded large amounts of solubleFaeG proteins.20,21 Transgenic plants in which theFaeG was targeted to the apoplast or ER showedonly marginal amounts of FaeG (Figure 1). The totalsoluble protein fraction of tobacco plant leafs of thebest transgenic lines obtained with apoplast-(clone6.5) and ER-targeting (clone 7.9) contained 0.025%and 0.015% of FaeG, respectively. In plants withchloroplast-targeting (clone 60.21), FaeG accumu-lated to up to 1% of the total soluble protein fraction.However, the levels of faeG-specific RNA weresimilar in plants targeting FaeG to the apoplast(clone 6.5), to the ER (clone 7.9) or to the chloroplasts(clone 60.21) (Figure 1). This suggests that the yield ofFaeG in the apoplast or ER is not limited bytranscription or mRNA stability but rather bytranslation, post-translational modification, translo-cation, or FaeG folding and stability at the destina-tion site. The total soluble protein fraction of leavesfrom plants transformed with apoplast-targeting orER-targeting FaeG constructs shows a heteroge-neous pattern of FaeG bands with a lower mobilitythan their chloroplast-targeted counterpart. Mostprobably this was due to N-glycosylation, as was thecase when FaeG was expressed in the endoplasmicreticulum (ER) of barley endosperm.19 Whether thepoor accumulation of FaeG in the apoplast and theER of tobacco leaveswas due toN-glycosylationwasnot studied.

Purification of chloroplast-targeted FaeG

We used a straightforward approach to purify thechloroplast-targeted FaeG from tobacco plant leaftissue without additional tags, based on the rela-tively high resistance of FaeG to pepsin digestion(Figure 2). Treatment with pepsin for 5 min wasoptimal to digest most plant proteins without adecrease in FaeG quantity. Longer digestion reducedthe amount of FaeG gradually. The residual con-taminating plant proteins and pepsin were removedin a two-step purification, to obtain more than 95%pure FaeG (Figure 2).

Crystal structure of the chloroplast-targetedFaeG

Both FaeG5/95 and FaeGGIS26 were assembled inthe chloroplast into strand-swapped dimers (Figure3(a); crystallographic details and model quality aresummarized in Tables 1 and 2). Both monomerstructures superimposed with a root-mean-squaredeviation of 0.3 Å. The chloroplast-targeted FaeGprotein has an Ig core made up of strands A1, A2, B1,B2, C, D, E1, E2, F and G, named according to theirplace in the Ig-fold and in analogy to the nomen-clature used for other fimbrial subunit structures.These show an incomplete Ig-like structure, lackingthe last β-strand G. In pili, this G-strand is provided

by the Nte of the adjacent subunit. In the chlor-oplast-targeted FaeG dimer, strand swapping occursbetween strands A1 and A2. As a result, the A1strand of monomer 1 is inserted in the Ig core ofmonomer 2, and the Nte of the chloroplast-targetedFaeG monomer becomes self-complementing.The Ig protein family shows a large degree of

variation. The Ig fold contains a common structuralcore of only four strands (B, C, E and F). Theaddition of three to five strands determines topolo-gical subclasses of this large protein family.19 In theIg fold of the chloroplast-targeted FaeG, a shorthelical turn occurs between strands A2 and B1, andan extra strand C′ is inserted between strands C andD. In addition to the Ig-folded core, the FaeGstructure contains an extra domain introducedbetween strands D and E1. This domain is com-posed of strands D′ and Dʺ, linked by two α-helices,α1 and α2.

An unswapped model of the FaeG structurediscloses details of the F4 fimbrial assembly

The domain-swapped dimeric structure of FaeG isincompatible with the donor strand complementa-

794 Crystal Structure of the F4 Fimbrial Subunit FaeG

tion/exchange in fimbrial assembly. We rearrangedthe FaeG structure in such a way that strand A1 isretained within the Ig core of one monomer. In the

resulting unswapped dimer, the monomers mu-tually donor strand complement each other's fold(Figure 3(b)). Although this unswapped dimerwould not be functional in fibre formation, theFaeG structure in the model is closer to the onepresumed to be present in F4 fimbriae, where thehydrophobic groove of one subunit is shielded bythe Nte of the adjacent subunit. The model predictsthat residues 1–17 form the Nte involved in thedonor strand exchange mechanism (Figure 3(c)).Residues 6–17 form the G-strand that aligns anti-parallel with the F strand of FaeG. In addition to 19main-chain hydrogen bonds, the Nte interacts withthe FaeG core through a pattern of alternatinghydrophobic residues. The side-chains of residuesPhe6, Val10, Ile12 and Ile16 are buried in the core ofFaeG. Moreover, burial of residue Trp1 in a shallowpocket on the surface of FaeG anchors the Nte in theFaeG core.

Chloroplast-targeted FaeG5/95 and FaeGGIS26show the same characteristics as their F4fimbrial counterparts

The solubility and stability of the chloroplast-targeted FaeG was unexpected. Intrinsically, theirincomplete fold makes fimbrial subunits unstableand susceptible to aggregation and/or proteolyticdegradation in the absence of a specific chaperone.4

Indeed, FaeG subunits are degraded upon periplas-mic expression in E. coli in the absence thechaperone FaeE,22,23 and FaeE deletion mutantsdo not accumulate FaeG.24,25 Therefore, fimbrialsubunits are typically co-expressed with their chap-erone,6–8 or as donor strand-complementedconstructs.10,26 Overproduction of recombinantFaeG in the cytoplasm of E. coli has been reportedto yield insoluble aggregates, which could besolubilized only by using SDS as a denaturant. Thisrefolded FaeG showed receptor binding capacityand was able to induce a mucosal immune responseupon oral administration to piglets. However, thestability of this SDS-refolded FaeG was low.27

F4ac fimbriae of ETEC strain 5/95 are less stablethan F4 fimbriae of other F4ac strains, including theGIS26 strain. Whereas non-boiled F4ac fimbriae

Figure 3. Three-dimensional structure of the chloro-plast-targeted FaeG. (a) Ribbon presentation and topologydiagram of the crystal structure of the chloroplast-targetedFaeG. The molecules are differentiated by colour, topologyis annotated to the cyan molecule. The N-terminal MDRSsequence resulting from the cloning of the chloroplast-targeted FaeG did not interfere with crystallization oraffect the dimerisation of FaeG. Only the serine residuewas visible in the Fo–2Fc map. (b) Ribbon presentation andtopology diagram of the unswapped dimer model ofFaeG. Colour and annotations are as in (a). (c) Close-upview of the interaction between the Nte (ball and stickpresentation) and the FaeG hydrophobic groove (yellow)in the model of the unswapped dimer. Amino acids areidentified by the single-letter code. (b) and (c) ResiduesThr31 and Gly32 are missing (broken line in the topologydiagram).

Table 1. Data-collection and crystal parameters for the FaeG5/95 and FaeGGIS26 crystals

FaeG5/95native

FaeG5/95 MAD dataset on Br soaked crystal

FaeGGIS26Absorption edge Inflection point High-energy remote

Wavelength (Å) 0.8123 0.9189 0.9195 0.9161 0.8075Space group C2 C2 P212121Unit cell dimensions

a (Å) 77.3 76.9 56.2b (Å) 57.6 57.6 91.2c (Å) 69.9 69.7 108.4

α, β, γ (deg.) β=112.5 β=112.2Resolution range (Å) 50–1.55 15–1.80 15–1.76 15–1.80 35–1.9Total reflections 418,795 523,615 514,317 560,121 460,525Unique reflections 41,286 26,408 31,586 26,565 44,786Redundancy 4.2 (4.0) 7.6 (7.3) 7.6 (6.3) 7.6 (7.1) 6.1 (6.1)<I/σI> 11.1 (3.33) 22.62 (4.92) 21.9 (4.73) 20.69 (3.80) 14.25 (3.77)Completeness (%) 99.8 (99.3) 99.9 (98.9) 84.2 (40.5) 99.6 (95.6) 99.9 (100)Rmerge (%) 5.0 (3.9) 10.0 (43.6) 9.8 (47.2) 11.0 (54.9) 9.4 (44.8)Ranomalous (%) 4.9 (17.6) 4.5 (18.2) 5.3 (22.7)Mosaicity (deg.) 0.979 0.72 0.373Beamline X11 BW7A X13R-factor (%) 18.4 18.3Rfree 20.5 22.6

Rmerge=Σ|I –<I>|/ΣI, where I is observed intensity, and <I> is the average intensity for symmetry.

795Crystal Structure of the F4 Fimbrial Subunit FaeG

show a characteristic ladder of FaeG polymers onSDS-PAGE, as detected by FaeG immunoblotting,non-boiled F4ac5/95 appear only as monomers.17Interestingly, the chloroplast-targeted FaeG5/95 andFaeGGIS26 proteins showed the same behavior astheir F4ac fimbrial counterparts. Non-boiled FaeG5/95appeared only as monomers, whereas FaeGGIS26dimers were detected by FaeG immunoblotting(Figure 4). This suggests that the FaeG5/95 subunitis less stable than the FaeGGIS26 subunit. Moreover,mapping of the seven amino acid differencesbetween FaeG5/95 and FaeGGIS26 on a model (Figure5) of F4ac fimbriae based on the unswapped FaeGstructure does not suggest any influence of theseresidues on polymer stability.Oral immunization with purified F4acGIS26 fim-

briae reduces ETEC excretion following challengemore efficiently than F4ac5/95.

18,20,27 The greaterstability of FaeGGIS26 could thus play a role in theimmunogenicity. It has been shown that chloroplast-targeted FaeG5/95 has a F4ac receptor-bindingcapacity and elicits F4ac-specific mucosal immuneresponse upon oral administration.20,21 Whether the

Table 2. Quality parameters for the final models of thechloroplast-targeted FaeG5/95 and FaeGGIS26

FaeG5/95 FaeGGIS26

Distribution of the residues in the Ramachandran plotMost favoured zones (%) 88.8 89.7Allowed zones (%) 11.2 10.3Generously allowed zones (%) 0.0 0.0Disallowed zones (%) 0.0 0.0

r.m.s.d. from idealBond lengths (Å) 0.009 0.014Bond angles (deg.) 1.228 1.426

Rcryst (%) 18.5 18.5Rfree (%) 20.5 22.6

chloroplast-targeted FaeGGIS26 is more immuno-genic than its FaeG5/95 counterpart warrantsanother study.

The FaeG structure indicates the F4receptor-binding domain

Although several studies have indicated glyco-proteins and/or glycolipids as the F4 receptor, thecarbohydrate moiety to which FaeG binds has notbeen identified.28–32 Conserved as well as variableepitope regions of FaeG have been proposed to beinvolved in F4 receptor binding.9 The oligopeptidesSer148-Leu-Phe150 and Ala156-Ile-Phe158 inhibitedhemagglutination by F4ab, F4ac and F4ad fimbriaeand binding of F4ab to epithelial brush borders. Thesubstitution of Phe150 by serine abolished F4abhemagglutination.33,34 Bakker et al. described thereceptor binding site on FaeG as a spatial arrange-ment of two amino acid residues with a hydrophobicside-chain (Phe/Leu134 and Phe/Leu/Met147) incombination with one or more amino acid residueswith hydrophilic and charged side-chains (Lys/Arg136, Arg/Ser/His155 and Asp/Asn216, or in thehypervariable region comprised of residues 163–173).9The structure of FaeG allows us to localize the

aforementioned residues and the hypervariableregions comprising residues 163–173 and residues206–21618 on the surface of FaeG (Figure 5). Thissuggests that the receptor-binding site of FaeG is notlocated within the Ig core of the protein but rather inthe extra domain made up of strands D′ and Dʺ, andα-helices α1 and α2. Especially the long loopbetween D′ and α1 and the loop connecting theextra domain to the Ig core (between Dʺ and E1) areindicated as being part of the binding site. Similarly,the receptor-binding site of the PapGII tip-adhesin

Figure 4. Comparison of the tobacco plant chloro-plast-targeted FaeG5/95 and FaeGGIS26 with their F4fimbrial counterparts by staining with Coomassie brilliantblue (a) and FaeG immunoblotting (b). Lane 1, totalsoluble protein extract from tobacco plants expressingFaeGGIS26; lane 2, total soluble protein extract from plantsexpressing FaeG5/95; lane 3, purified F4acGIS26 fimbriae;lane 4, purified F4ac5/95 fimbriae. Samples were separatedby SDS-PAGE (10% (w/v) polyacrylamide gel) with orwithout prior boiling in SDS loading buffer.

796 Crystal Structure of the F4 Fimbrial Subunit FaeG

has been shown to be located outside the Ig core ofthe lectin domain.35

Figure 5. Model of F4 fimbriae, based on the structureof FaeG. The molecules were oriented in such a way thatthe fimbriae are approximately 4 nm wide, as wasobserved by electron microscopy.51 Molecules arecoloured alternately in cyan and green,. Red residueshave been proposed to be involved in receptor binding.Blue residues differ in the FaeG sequence of ETEC strains5/95 and GIS26. The inset shows a close-up view in aribbon presentation of the residues suggested to beinvolved in receptor binding.

Conclusions

The ability of tobacco chloroplasts to producestable and soluble FaeG was unforeseen, sincefimbrial subunits have been shown to be susceptibleto aggregation and/or proteolytic degradation inthe absence of a specific chaperone.4 Interestingly,the strand-swapping between strands A1 and A2seen in the dimeric structure of the chloroplast-targeted FaeG, occurred in the cytoplasm of E. coliwhen the subunits of the Dr fimbrial family wereexpressed in the absence of their chaperone.11,12,36

During donor strand complementation, capping ofthe hydrophobic groove on the surface of thesubunit by the chaperone is concerted with thefolding of the subunit.37 A possible role of the donorstrand of the chaperone could be to keep the A1 andA2 strands within the Ig core. The chaperone wouldthereby prevent the subunits from folding into an

assembly-incompetent form before in vivo fimbrialassembly.

Materials and Methods

Expression of FaeG in different tobacco plant cellcompartments

In order to target FaeG to different plant compartments,the FaeG-encoding gene from ETEC 5/95 (AY437806) andGIS26 (AJ616236) without signal sequence was fusedeither to the chloroplast transit peptide (TP, FaeG5/95 andFaeGGIS26) of the pea rubisco small subunit (J01257) or theapoplast-targeting signal sequence (SS, only FaeG5/95)from barley trypsin inhibitor (X65875). To retain FaeG inthe ER, the hexapeptide SEKDEL was added to the Cterminus of SS-FaeG (Figure 1). The gene cassettes wereintroduced to binary or co-integrative plant expressionvectors,38,39 and conjugated into the rifampicin-resistantAgrobacterium tumefaciens strain C58C140 containing themodified Ti plasmid pGV226041 using triparentalmating.42 Thereafter, transgenic tobacco (Nicotiana taba-cum L.) plants were obtained by leaf disk co-cultivation.43

Leaves from ten independent, one month old transfor-mants were frozen in liquid nitrogen and stored at −70 °Cfor DNA, RNA and protein analysis. DNA hybridizationand protein analyses were performed as described.20,21

Total RNAwas prepared using the RNeasy Plant Mini kit(Qiagen) and separated in formaldehyde gels, andhybridized as described.20

797Crystal Structure of the F4 Fimbrial Subunit FaeG

Purification of F4 fimbriae from ETEC and FaeG fromtobacco leaves

F4ac fimbriae were purified as described.18 The totalsoluble protein fraction was extracted from transgenictobacco plants expressing FaeG as described.21 To purifyFaeG, this extract was exposed to digestion by pepsin(0.16% (w/v)) at pH 3.5 for 5 min at 37 °C. The digestionwas quenched by raising the pH to 8 by adding Na2CO3 toa final concentration of 45 mM. After centrifugation for20 min at 4 °C at 10,000g, the supernatant was subjected toprecipitation in 32% (w/v) (NH4)2SO4 on ice for 2 h. Theprecipitated proteins were dissolved in PBS (8.1 mMNa2HPO4, 1.8 mM KH2PO4, pH 7.5, 137 mM NaCl,2.7 mM KCl). Upon dialysis against PBS, the sample wasloaded onto a Q-Sepharose column (Amersham Bios-ciences) equilibrated in 20 mM phosphate buffer (pH 7.4).FaeG was eluted by increasing the concentration of NaClin the buffer. Fractions containing FaeG were pooled,dialyzed to PBS and concentrated to 10 mg/ml, and usedas such for crystallization.

Crystallization and X-ray data collection

All crystals were grown using the hanging-drop,vapour-diffusion method at 293K: the FaeG5/95 crystalsused for the native dataset in 0.1M sodium cacodylate (pH6.5), 1.4 M sodium acetate trihydrate; the FaeG5/95 crystalsused for NaBr soaking in 2.0 M (NH4)2SO4, 0.1 M Tris (pH8.5); the FaeGGIS26 crystals in 30% (w/v) PEG 8000, 0.1 Msodium cacodylate (pH 6.5), 0.2 M NH4H2PO4. X-ray datawere collected at the DESY/EMBL beamlines BW7A, X11,and X13 (Hamburg, Germany). Data for the NaBr-soakedFaeG5/95 crystal were collected at the absorption edge,inflection point and high-energy remote wavelengths asdetermined by a fluorescence scan for Br–. Data wereindexed and processed using DENZO and SCALEPACKfrom the HKL suite.44 TRUNCATE from the CCP4 suite45

was used to obtain structure factor amplitudes. Prelimin-ary phases from the MAD data were calculated at theEMBL facility using the experimental Auto-Rickshawautomated crystal structure determination platform.46

The initial model for FaeG5/95 was built from the nativeand MAD datasets with the advanced version of Auto-Rickshaw using the programs MLPHARE and DM fromthe CCP4 suite,45 and ARP/wARP.47 The initial FaeGGIS26model was obtained by molecular replacement with theFaeG5/95 model, using Phaser.48 The models werecompleted by manual model building in Coot49 andrefined using Refmac5.50

Protein Data Bank accession numbers

Coordinates for the chloroplast-targeted FaeG5/95 andFaeGGIS26 structures have been submitted to the ProteinData Bank with accession numbers 2J6G and 2J6R.

Acknowledgements

The Federation of European MicrobiologicalSocieties is acknowledged for providing a researchfellowship for J.J.J. J.B. and L.B. are postdoctoralfellows of the Fonds voor Wetenschappelijk Onder-

zoek–Vlaanderen (FWO-Vlaanderen), which alsogranted financial support (FWO- G.0513.04) and theDNA sequencing equipment (FWOAL215). We aregrateful to the EMBL for the use of the beamlines X11,X13 and BW7A at the DESY synchrotron, Hamburg.

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Edited by J. Karn

(Received 5 October 2006; received in revised form 7 February 2007; accepted 12 February 2007)Available online 22 February 2007