Supporting InformationTanabe et al. 10.1073/pnas.0912115107SI TextDiscussion Structural comparison between N. meningitidis PorB andGram-negative porins. PorB was previously predicted to sharethe stable, 16-stranded β-barrel scaffold with other porins, andthe C. acidovorans Omp32 [PDBID; 1E54 (1)] has been usedas a template for homology modeling (2). In this study, bothOmpC (3) and Omp32 (1) were used as molecular replacementsearch models using PHASER (4), MOLREP (5) and AMORE(6), however, even extensively modified search models did notresult in a reasonable solution. Nevertheless, superposition ofPorB with the structure of the E. coli OmpF [PDBID; 2OMF(7)], the structure of the E. coli OmpC [PDBID; 2J1N (7)],and the structure of the C. acidovorans Omp32 [PDBID;1E54(1)] resulted rms deviation values for the Cα atoms of 2.0, 1.8,and 1.8 Å, respectively. The majority of the sequence and struc-tural similarity is, as anticipated, within the transmembranestrands of the β-barrel (Fig. S3 A–D). In a sequence alignment,however, only 20 absolutely conserved residues are identified.These absolutely conserved residues fall into three main cate-gories: aromatics, glycines, and charged residues within the pore(Fig. S3 E and F).

As has previously been noted for other porins (8) rings of aro-matic amino acids are present on the periphery of the β-barrel inPorB and delineate the membrane boundaries, and 6 of the ab-solutely conserved residues in the multiple porin alignment arearomatics that function in this manner. However, the moststrongly conserved residues amongst the porins in this analysis areglycines (7 out of 20 amino acids; Fig. S3 E and F). Two of these,G88 and G143, are located in tight turns, and five (G6, G44, G47,G93, G336) are located at the trimer interface. The expanded setof Ramachandran angles available to glycine residues may sup-port their location in the turns, whereas packing considerationslikely explain their prevalence in the trimer interface.

The third subset of conserved residues is located along the an-ion translocation pathway within the pore. A cluster of lysines andarginines (equivalent to PorB K42, R77, and R130) and a gluta-mate (equivalent to PorB E62) emanate from the barrel wall toform a structurally conserved permeation pathway in each porin.The similarity implies that the basic mechanism of anion trans-port in these porins is likely shared.

Interestingly, the intracellular helix of the L3 loop is also struc-turally conserved (Fig. S3C) despite low sequence identity in thisregion (Fig. S3D). The L3 loop has been extensively mutagenizedin both OmpF and OmpC, and the function of these mutants hasbeen probed by labeling and electrophysiological measurements.As a summary of these extensive studies, the charged amino acidsof L3 are likely to be important for channel selectivity (9–12) butthere is controversy on the role of this loop in voltage gating(9, 11).

In comparison, the functions of the other extracellular loopsthat decorate the β-barrel scaffold of porins have not been as wellstudied. These loops lack appreciable sequence and their diver-gent evolution may modulate channel function to make each por-in unique.

Structural determinants of the pNTP binding site.The location of thepNTP binding site within the pore is at a region of high sequenceand structural conservation in neiserial and other Gram-negativebacterial porins. The protein side chains that likely stabilizeAMP–PNP binding within the PorB pore (Lys 9, Lys 42, Arg77, Asn 96, Lys 100, and Arg 130) are all absolutely or highly con-served (i.e., Lys to Arg substitutions) in PorB homologs from both

pathogenic and commensal neissera (Fig. S2) despite the latterhaving been shown not to bind to pNTP (13). In addition, threeof the six side chains (Lys 42, Arg 77, and Arg 130) are highlyconserved between PorB and other Gram-negative porins. Bind-ing specificity for ATP may additionally result from a shift of theposition of the intracellular helix of L3 in PorB as compared toother porins. This shift arranges the conserved amino acid sidechains in an improved orientation for pNTP binding. The struc-tural origins of this helix shift are complex and likely require nu-merous substitutions throughout the protein.

An unusual aspect of the AMP–PNP binding site in PorB is theabsence of divalent cation associated with the nucleotide. Otherthan PorB, only 2 protein structures in the PDB bind to isolatedpurine nucleotide without a divalent cation (most commonlyMg2þ): the CBS domain of the CLC chloride channel (14) andthe adenylate isopentenyltransferase from Humulus lupulus(15). One role of the divalent cation includes stabilization of thenegative charge from the β- and γ-phosphates (or phosphate ana-logs), which lowers the free energy of ATP hydrolysis by an orderof magnitude. In PorB, the side chains of Lys 42 and Asn 96 formdirect hydrogen-bonding contacts to the β- and γ-phosphates, re-spectively, potentially substituting for Mg2þ. AlthoughMg-ATP isthe physiologically relevant species in the mitochondria, inclusionof Mg2þ in the cocrystallization experiment dissolved the PorBcrystals, and previous electrophysiological analysis did not re-quire Mg2þ to alter channel gating.

The N. meningitidis PorB does not exhibit nucleotide triphosphate hy-drolysis activity. Both ATP and GTP have been reported to bindPorB and modulate channel conductance (13). Physiologically,purine nucleotide triphosphate (pNTP) regulation of PorB mostlikely occurs when PorB is inserted into the mitochondrial outermembrane, because ATP is available. Six independent diffractiondatasets of PorB soaked in 1–5 mM ATP with and without Mg2þfor 1–3 d and five independent datasets of PorB soaked with1–5 mM GTP with and without Mg2þ for 1–3 d were collected,yet no appreciable density was observed for pNTP in any of thesedatasets. By comparison, every diffraction dataset of PorB soakedwith AMP–PNP showed strong electron density corresponding tothis nonhydrolyzable nucleotide analog. As a result, the pNTPhydrolysis activity was assessed. pNTP hydrolysis was measuredby the LeBel method, which quantitates released phosphate(16). Based on the lack of pNTP hydrolysis observed by this meth-od (Fig. S6), it is possible that the AMP–PNP simply binds moretightly to PorB in the crystallization conditions.

Stoichiometry of the TLR2-PorB complex. To provide a qualitativeassessment that the sTLR2 and PorB form a direct complex, hu-man hexahistidine-tagged sTLR2 and N. meningitidis PorB withno histidine tag were first purified separately. Complex formationbetween purified sTLR2 and PorB was then evaluated by prein-cubation of PorB and sTLR2 for 4 hours and coinjection onto asize exclusion column in 50 mM Hepes pH 7.5, 100 mM NaCl,0.1% LDAO. Analysis was performed in quadruplicate usingtwo different gel filtration columns and two different Akta Puri-fier systems. The resultant chromatograms all revealed the for-mation a monodisperse species with an approximate molecularweight of 405 kD (Fig. S7A). To assess whether electrostatic at-traction contributes to the formation of this complex, size exclu-sion chromatography of coinjected sTRL2 and N. meningitidisPorB was repeated in triplicate in the same buffer containing300 mM NaCl (Fig. S7B). The decrease in complex formation

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is consistent with a mechanism where electrostatics contribute tothe formation of the complex. As controls, samples containingeither sTLR2 and chicken albumin in 0.1% LDAO or PorBand chicken albumin in 0.1% LDAO were coinjected; neitherprotein showed nonspecific interactions nor aggregation underthese conditions (Fig. S7C).

The molecular weights of the proteins that coeluted as a405 kD complex on size exclusion chromatography were evalu-ated by SDS-PAGE (Fig. S7D) following additional purificationon a Co2þ affinity Hi-trap column equilibrated in 20 mM Tris-HCl pH 7.5, 100 mM NaCl, and 0.1% LDAO. The beads werewashed in the same buffer containing 100 mM imidazole andeluted with the same buffer containing 500 mM imidazole.SDS-PAGE evaluation shows bands consistent with both sTLR2and PorB, suggesting these two form a direct complex (Fig. S7D).

PorB is a physiological trimer of 114 kD that has an apparentmolecular weight of 150 kD when run on size exclusion column,likely as a result of the presence of the detergent micelle. Theectodomain of TLR2 is a 65 kD monomer. Physiologically, itforms a heterodimer with TLR1, and in this context likely bindsas a 130 kD dimer (17, 18). To identify the stoichiometry of the405 kD sTLR2-PorB complex, the contribution of each of theseproteins and detergent to the complex was measured. The deter-gent contribution for each PorB trimer was measured in quintu-plicate using MultiAngle Laser Light Scattering (Dawn Heleos 8Wyatt tech) with a 658 nm wavelength in 50 mM Hepes-NaOHpH 7.5, 100 mM NaCl with 0.1% LDAO. Analysis using ASTRAversion 5.3.4.14 identified that the detergent-solubilized PorB tri-mer had a mass of 146� 4 kD, with the mass of the protein con-tributing 121 kD� 4 kD and the mass of the LDAO contributing25 kD� 2 kD in close agreement with the published value forLDAO micelle size (ref. 19 and Fig. S7E). Taken together, themolecular weight of the complex agrees best with a 2∶6 ratioof sTLR2 to PorB. This stoichiometry is consistent with knownoligomerization states because sTLR2 is likely a dimer, and PorBis a physiological trimer.

Materials and Methods Crystallization and structure determination.Cloning, purification, and crystallization of PorB were performedas described (20). Briefly, the gene encoding the patient isolate ofN. meningitidis PorB (21) was cloned into the pET21 vector andtransformed into E. coli BL21(DE3) cells lacking several endog-enous porins (22). For native protein, the cells were grown in LB-medium, whereas for Se-Met incorporated PorB the cells weregrown in minimal medium supplemented with 25 mg∕L sele-no-L-methionine, as described (23). The cells were grown at30 °C until the optical density measured at 600 nm (OD600)reached 0.5. At this point, expression was induced with0.3 mM IPTG, and the protein was expressed at 37 °C for 4 hours.Recombinant PorB expressed into inclusion bodies. The inclusionbodies were washed three times in buffer containing 20 mM Tris-HCl, 200 mM NaCl, 1 mM EDTA, and 2% Triton X-100, solu-bilized in 1 mL of denaturing buffer [50 mM Tris-HCl (pH 7.5),200 mM NaCl, 7.2 M urea], and then quickly mixed with 2 mL ofrefolding buffer [50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 10%LDAO (Lauryl dimethyl amine-N-oxide)]. Refolded PorB was se-parated from aggregates using a Hi-load Superdex 200 16∕60 sizeexclusion column equilibrated with 50 mM Tris-HCl (pH 7.5),200 mM NaCl, 1 mM EDTA and 0.15% LDAO.

PorB was concentrated to 15 mg∕mL in 20 mM Tris-HCl pH7.5, 200 mM NaCl and 0.01% (w∕v) n-Tetradecyl-N,N,dimethyl-glycine (TDDG) prior to crystallization trials. During this con-centration step, the detergent was exchanged from LDAO toTDDG by three serial 1∶100 dilutions in TDDG-containing buf-fer. Initial crystals of PorB were identified from a 1440 conditionsparse matrix screen. Crystals were optimized using diffraction-based feedback at the Stanford Synchrotron Radiation Light-source (SSRL) beamlines 7-1, 9-1, 9-2, 11-1, and 12-2, the Ad-

vanced Light Source (ALS) beamlines 8.2.2 and 8.3.1, theAdvanced Photon Source (APS) Life Sciences Collaborative Ac-cess Team (LS-CAT) beamlines, and the Cornell High EnergySynchrotron Source (CHESS) beamlines A1 and F2. Optimizedcrystals were grown by the hanging drop vapor diffusion methodfrom PorB at 15 mg∕mL in 0.1% TDDG detergent equilibratedagainst mother liquor containing 100 mM MES pH 6.0–6.5,50 mM CsCl, and 28–32% (w∕v) Jeffamine M-600. Prior to datacollection, crystals were quickly dragged through a crystallizationsolution with the concentration of Jeffamine M-600 increased to35–40% (w∕v) for cryoprotection, followed by flash-cooling in li-quid nitrogen. Approximately 95% of the crystals belonged to therhombohedral space group R32 with a ¼ b ¼ 148 Å c ¼ 112 Å.These rhombohedral crystals grew to a maximal size of 0.10×0.10 × 0.02 mm. Less than 5% of native crystals formed in thehexagonal space group P63 with unit cell dimensions a ¼ b ¼83 Å, c ¼ 106 Å. However, Se-Met incorporated PorB formedcrystals exclusively in the hexagonal P63 space group. The hexa-gonal crystals formed within 2–3 months to a maximal sizeof 0.05 × 0.05 × 0.1 mm.

Because of the superior diffraction quality of the P63 crystalsand the better reproducibility of Se-Met incorporated PorB form-ing crystals in the P63 space group, all heavy atom derivatizationwas performed on crystals of Se-Met incorporated protein. TheLu3þ Er3þ and WO2−

4 derivatives were prepared by soaking Se-Met PorB crystals with 1 mM LuðCH3COOÞ3 for 3 hours, 1 mMNa2WO4 for 3 hours, or 5 mM ErðCH3COOÞ3 for 3 d. Data werecollected using wavelengths and beamlines listed in Table S2. Thelocation of the position of the Lu3þ was identified using theSHELXD (24) subroutine in the program SHARP (25). Sitesfor the other two heavy atoms and the Se-Met locations wereidentified using difference Fourier maps phased with the Lu3þderivative. Heavy atom positions were refined and the phasingcalculation was performed using the program SHARP. Thephases from SHARP were dramatically improved using solventflattening with histogram matching in RESOLVE (26) andARP/wARP (27) and multiple crystal form averaging betweenthe R32 and P63 crystal forms in DMMULTI (28, 29).

All data were processed and scaled using the HKL (30) andCCP4 suites of programs (28). Manual model building was per-formed using O (31) and COOT (32) and refinement was per-formed using CNS (33) and REFMAC (28, 34). The sequencealignment and superposition of PorB with Gram-negative porinswere performed by ESPript (35) and SSM superpose (36) in Coot(32). Crystallization of PorB with small molecule ligands was per-formed a minimum of three times by both soaking and cocrystal-lization methods using standard crystallization conditions andadding 1–10 mM ATP, 1–10 mM AMP–PNP, 1–5% stachyose,1–5% raffinose, 1–5% sucrose, 1–5% galactose, 1–5% arabinose,or 1–5% glucose. Of these trials, electron density was observedfor AMP–PNP, sucrose, and galactose. Parameter and topologyfiles for ligands were generated using PRODRG (37). The geo-metry of each final model was assessed using PROCHECK (38).Each refined model has all residues in the most favored or ad-ditionally allowed regions of the Ramachandran diagram. Therefinement statistics for all models are shown in Table S1. Elec-trostatic potentials were calculated at a temperature of 310 Kusing the nonlinear Poisson–Boltzmann equation, as implemen-ted in APBS version 4.0 (39). Figures were prepared using Py-Mol (40).

ATP hydrolysis assays. ATP hydrolysis was performed in a 0.5 mlvolume containing one of three buffers: a standard assay buffer(20 mM Tris-HCl pH 7.5, 200 mM NaCl, 2 mM ATP or 2 mMGTP, 2 mM MgCl2, and 10 mM KCl); a low pH buffer(100 mM MES pH 6.0, 200 mM NaCl, 50 mM CsCl, 2 mMATP or 2 mM GTP, 2 mM MgCl2, and 10 mM KCl); or a buffermimicking the crystallization conditions (100 mM MES pH 6.0,

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200 mM NaCl, 50 mM CsCl, 2 mM ATP or 2 mM GTP). Reac-tions were initiated by the addition of 10 μL of 1 mg∕mL purifiedPorB. The standard and low pH reactions were performed at 37 °C for 20 min. The reactions performed in the crystallization buf-fer were performed at 18 °C for 3 d, which mimics the crystalliza-tion conditions. The reaction was terminated by addition of0.5 mL ice-cold 0.5 M trichloroacetic acid. After addition of0.5 mL of LeBel Reagent, the sample was filtered to remove in-soluble material, and the OD745 was measured. Naþ-Kþ ATPaseisolated from porcine cerebral cortex (Sigma), and wild-type Gαiwere used as positive controls.

Expression and purification of the TLR2 ectodomain (sTLR2). ThecDNA encoding the ectodomain of TLR2 (sTLR2) was sub-

cloned into the pPICZα-B vector under the control of theAOX1 promoter in the pPICZα-B vector (Invitrogen). P. pastorisX-33 cells harboring sTLR2-pPICZα-B were grown for 18 hoursuntil OD600 ¼ 2.0–2.5 and were induced by transferring the cellsto medium supplemented with 0.5% methanol. After 24 h, cellswere harvested by centrifugation at 4000 × g for 15 min. Thecells were disrupted using a Bead-beater in the presence of0.5 mm glass beads. The lysate was clarified by centrifugationat 30; 000 × g for 1 hour, then the supernatant was loaded ontoa Co2þ-Hi-trap column. sTLR2 was eluted using 100 mM imid-azole. sTLR2 was verified to be monodisperse and pure by sizeexclusion chromatography and Coomassie stained SDS-PAGE.sTLR2 was unstable in the absence of 100 mM NaCl.

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Fig. S1. Representative electron density maps for PorB. (A) Experimental electron density for the PorB trimer. (B) Experimental density for a representative β-strand of the β-barrel fold. (C) 2jFoj − jFcj density calculated using phases from the refined model (PDBID:3A2R) and corresponding to the same region as B.

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Fig. S2. Topology and sequence analysis of PorB. (A) Topology diagram of PorB. β-strands are shown with cyan arrows whereas α-helices are shown with redcylinders. (B) Sequence alignment of the N. meningitidis PorB (pathogen), N. gonorrhoeae PorB (pathogen), N. lactatemia PorB (commensal), and N. mucosaPorB (commensal) with the secondary structural elements of PorB. β-strands are shown with cyan arrows whereas α-helices are shown with red cylinders. Thesequence alignment was performed by ClustalW (1) using the PIB sequence characterized by Gotschlich et al. (2) and the sequence of the PorB W135 variantused for this study (21). Absolutely conserved residues are highlighted with bold text, whereas regions of relatively high conservations are delineated with grayshading. Residues involved in sugar binding (T7, K9, K42, E110) are colored blue. Residues likely involved in AMP–PNP binding (K9, K42, K62, R77, N96, K100,R130) are colored red. K9 and K42 are involved in both sugar and AMP–PNP binding and is colored magenta. The penB antibiotic resistance mutations in N.gonorrhoeae (equivalent to G103, D104) are underlined. There are over 100 sequence variations of N. meningitidis PorB reported in the literature, with dif-ferences mapping to the interstrand loops. This study used a strain W135 cloned from a patient isolate that contained two amino acid substitutions as com-pared to the reported sequence (3). Positions where the patient isolate differ from the published sequence map to L4 and L6, and are highlighted orange. Foran alignment of all currently known N. meningitidis PorB sequences, see http://neisseria.org/nm/typing/porb/. (C) The structure of PorB illustrated as a cartoonwith β-strands in cyan α-helices in red, and loops in gray.

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Fig. S3. Comparison of PorB and other Gram-negative porins. (A)–(C) Overlay of PorB, Omp32, OmpC, and OmpF. Secondary structural elements common to allfour porins are shown in gray, whereas regions where the proteins differ in structure are shownwith color. Elements unique to PorB are shown in red, elementsunique to Omp32 are shown in blue, elements unique to OmpC are shown in green, and elements unique to OmpF are shown in yellow. (D) Sequence con-servation of Gram-negative porins superimposed on the PorB trimer. The secondary structure of PorB is colored from no sequence identity (Gray) to 50%identical (White) to 75% identical (Orange) to 100% identical. Positions where the sequence is absolutely conserved additionally show the side chain high-lighted with a cpk representation. (E) Structure-based sequence alignment of PorB with Gram-negative porins. The secondary structure elements for PorB areincluded for orientation. (F) Location of conserved glycine residues superimposed onto the PorB trimer.

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Fig. S4. A positively charged cage facilitates sugar binding in outer membrane proteins. (A, B) Costructure of PorB with sucrose (PDBID 3A2S). Note theasymmetric binding of sucrose to one face of the pore. (C, D) Costructure of the Salmonella typhimurium ScrY with sucrose [PDBID 1A0T; (1)] and (E, F) Cost-ructure of the E. coli LamB with sucrose [PDBID 1AF6; (2)]. The blue rectangles in (B, D, F) are delineated by Arginine and Lysine residues at each corner (RKcage), forming a positively charged cage for sugar binding. The dimensions of the cage are ∼11–12 Å × ∼6–8 Å.

1. Forst D, Welte W, Wacker T, Diederichs K (1998) Structure of the sucrose-specific porin ScrY from Salmonella typhimurium and its complex with sucrose. Nat Struct Biol 5:37–46.2. Wang YF, et al. (1997) Channel specificity: Structural basis for sugar discrimination and differential flux rates in maltoporin. J Mol Biol 272:56–63.

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Fig. S5. Identification of a putative nonselective translocation pathway through PorB. (A) and (B) Locations of the Er3þ (Orange) and Lu3þ (Blue) ions in theheavy atom derivative datasets of PorB suggests that these bind to PorB using water-mediated contacts to negatively charged side chains and backbonecarbonyls approaching the pore. The view is (A) from the extracellular space and (B) rotated 90° and viewed through the membrane. Distances betweentrivalent heavy atoms and the protein are too long for direct coordination. (C) Distances between the protein and Lu3þ are all >3.4 Å. Lu3þ has an idealcoordination distance of 2.4 Å. (D) Distances between PorB and Er3þ are all >3.2 Å. Er3þ has an ideal coordination distance of 2.3 Å. (E) and (F) The locationof theN. gonorrhoeae PIB antibiotic resistance mutations corresponding to the PenBmutations have been mapped onto the PorB structure using the sequencealignment in Fig. S2B. These mutations (Gly103 and Asp104) are highlighted in yellow. (E) Viewed from the extracellular space and (F) rotated 90° and viewedthrough the membrane. The heavy atom binding locations and the antibiotic resistance mutations are immediately adjacent and likely form a contiguoustranslocation pathway.

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Fig. S6. Biochemical analyses of PorB in the presence of ATP. (A) Lack of pNTP hydrolysis in the N. meningitidis PorB. All assays were performed in triplicate in ahigh pH buffer (pH 7.5), low pH buffer (pH 6.0), or in a buffer similar to the PorB crystallization conditions (cry) using independently purified protein samples.Naþ, Kþ-ATPase, and Gαi subunits were used as positive controls in the assays (SI Methods). PorB lacks nucleotide hydrolysis activity in all conditions assessed. (B)and (C) ATP has little effect on sugar transport in liposomal swelling assays. The change in OD400 upon addition of 5 mM ATP or 5 mM AMP–PNP to theliposomes (measured as the ratio between the OD400 in the absence and in the presence of ATP or AMP–PNP) diluted in isoosmotic concentrations of glucose,galactose, arabinose, maltose, sucrose, or stachyose. No effect was observed for any of these 12 combinations. Representative traces for (B) galactose in thepresence of ATP and (C) sucrose in the presence of ATP are shown plotted as a function of time. Black squares plot the ATP effect on empty liposomes, whereascolored symbols plot independent measurements with PorB-containing liposomes.

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Fig. S7. Interaction between sTLR2 and N. meningitidis PorB. (A) Size exclusion chromatograms of isolated sTLR2 (Red; elution volume 15.4 mL, i.e. 65 kDmonomer) and isolated PorB (Black; elution volume 12.5 mL, i.e., 114 kD trimerþ 25 kDLDAO micelle) compared to the chromatogram of a 1∶1 sTLR2:PorBmixture (Blue). The 10.7 mL elution volume of the complex correlates to a molecular weight of 405 kD, which is consistent with a 6∶2 PorB:sTLR2 ratio, andsuggests that two PorB trimers interact with a sTLR2 dimer in the complex. (B) Size exclusion chromatogram of sTLR and PorB (Black) coinjected in the presenceof 300mMNaCl shows decreased complex formation (Blue). The elution volumes are statistically identical to those in panelA. (C) Size exclusion chromatogramsof negative controls. The blue trace shows the chromatogram of coinjected sTLR2 and chicken albumin in 20mM Tris-HCl pH 7.5, 0.1% LDAO, and 100 mMNaClwhereas the green trace shows the chromatogram of and PorB and chicken albumin in the same buffer. (D) SDS-PAGE analysis of the PorB-TLR2 complexfollowing size exclusion chromatography and Co2þ affinity purification. The SDS-PAGE analysis shows the presence of both PorB and sTLR2 in this fraction.(E) MALLS profile of the PorB trimer. The UV chromatogram (Black) is superimposed with the multiangle light scattering trace and the calculated molecularweight of each peak as determined by the light scattering is shown with a black line. A single UV peak corresponds to the PorB trimer, whereas two lightscattering peaks correspond to the PorB trimer and the excess LDAO micelles, which are efficiently separated from protein by this procedure. The micelles arecalculated to contribute 25 kD to the apparent molecular mass of PorB on the size exclusion column.

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Table S1. Data collection and refinement statistics

Native + sucrose + galactose +AMP–PNP

Data collectionBeamline APS ID21-G SSRL11-1 APS ID21-D SSRL11-1Wavelength 0.978 0.978 0.978 0.979Space group P63 P63 P63 P63Unit cell

dimensionsa, c (Å) 83.0, 106.3 82.7, 106.4 84.3, 105.9 84.4, 106.3Resolution (Å) 50-2.3 (2.36-

2.3)50-2.2 (2.26-

2.2)50-3.2 (3.28-

3.2)50-2.9 (2.97-

2.9)Rsym 6.2 (24.7) 6.7 (36.4) 8.4 (14.8) 7.7 (33.8)I∕σI 23.1 (3.8) 22.0 (2.9) 16.4 (5.9) 20.4 (3.6)Completeness

(%)92.0 (61.0) 99.4 (96.1) 91.7 (51.5) 90.5 (47.0)

Redundancy 6.1 (3.9) 5.6 (3.2) 5.6 (3.1) 7.4 (6.0)Unique

reflections16247 19952 6538 8668

RefinementRwork∕Rfree 22.4/25.9 20.9/25.5 23.1/26.5 25.1/29.0No. atomsProtein 2601 2593 2593 2593Ligand 0 23 12 31LDAO 0 12 0 0Water 53 168 0 0RMS deviationsBond lengths (Å) 0.014 0.010 0.008 0.009Bond angles (°) 1.51 1.22 1.60 2.03

Values in parentheses are for the highest-resolution shell.

Table S2. Derivative data collection and phasing statistics

Se-MetSe-Met crystal soaked

with LuðCH3COOÞ3Se-Met crystal soaked

with Na2WO4

Se-Met crystal soakedwith ErðCH3COOÞ3 R32 crystal form

Data collectionBeamline SSRL9-2 ALS 8.3.1 ALS 8.3.1 CHESS F2 SSRL 11-1Wavelength 0.978 1.314 0.978 1.29 0.979Unit Cell dimensionsa, c (Å) 84.7, 106.1 83.6, 106.1 84.6, 105.8 84.4, 106.6 148.1, 117.1Resolution (Å) 50-2.4(2.46-2.4) 50-3.5(3.59-3.5) 50-2.7(2.77-2.7) 50-3.3(3.38-3.3) 50-2.9(2.97-2.9)Rsym 6.7(21.5) 11.8(44.3) 8.8(31.0) 11.3(45.8) 7.5 (12.1)I∕σI 19.3(4.7) 14.5(5.8) 18.5(3.3) 11.5(3.8) 12.7 (3.4)Completeness (%) 94.0(63.1) 98.8(98.2) 93.0(60.2) 99.9(100) 92.1 (54.8)Redundancy 5.2(4.0) 7.8(7.6) 8.0(6.2) 6.2(6.1) 7.5 (5.3)Phasing power 0.2 0.8 0.5 0.6 n/a

All derivative data soaks were performed on Se-Met crystals in the P63 crystal form. The overall figure of merit calculated in SHARP (25) was 0.36 at 2.8 Åresolution. The overall figure of merit after multiple crystal form averaging and solvent flattening in DMMULTI (28, 29) was 0.39 at 2.7 Å resolution. Values inparentheses are for the highest-resolution shell.

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