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1 Supplementary Table 1: Data collection, phasing and refinement statistics ChbC/Ta 6 Br 12 Native ChbC Data collection Space group P P Cell dimensions a, c (Å) , , Resolution (Å) 4.5 ( )* 3.3 ( ) R sym or R merge (0.379) (0.702) I/σ(I) 23.5 (3.75) 65.4 (2.3) Completeness (%) 98.2 (87.6) 95.7 (91.4) Redundancy Refinement Resolution (Å) ( ) No. reflections (4978) Completeness (%) 95.0 (86.0) R work/ R free 22.8/26.4 No. atoms Protein Ligand/ion 419 Water 0 B-factors Protein Ligand/ion Water R.m.s deviations Bond lengths (Å) Bond angles ( ) *Highest resolution shell is shown in parenthesis. 1

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3 Supplementary Figure 1: Sequence alignment of bacterial ChbC proteins. Sequence alignment of ChbCs from Bacillus cereus (NP_ ), Bacillus amyloliquefaciens (YP_ ), Enterobacter cancerogenus (EFC ), Escherichia coli (YP_ ), Erysipelothrix rhusiopathiae (ZP_ ), Klebsiella pneumoniae (ZP_ ), Salmonella enterica (YP_ ), Serratia odorifera (ZP_ ), Vibrio cholerae (ZP_ ), and Vibrio fischeri (YP_ ). PtsG (NP_ ), which transports glucose, and MtlA (ZP_ ), which transports mannitol, are from E. coli and belong to the superfamily of Glc EIIC, but are in different subfamilies than ChbC. Cylinders and arrows above the alignment correspond to α-helices and β-strands, respectively, and are color-coded by the same scheme as Fig. 2a. The Glc superfamily signature sequence is marked with blue lines, residues that form hydrogen bonds with the bound chitobiose are marked with red circles, and Glu334 and His250 are highlighted in red. 3

4 Supplementary Figure 2: Purification and stability of ChbC. (a) Purification of ChbC. Lane 1, molecular weight standard; Lane 2, sample after cobalt affinity column; lane 3, the same as lane 2 after incubation with TEV protease to remove the decahistidine tag; lane 4, sample after further purification by gel filtration. (b-e) Analytical gel filtration profiles of ChbC in (b) n-dodecyl-β-d-maltoside, (c) n-decyl-β-d-maltoside, (d) n-nonyl-β-d-maltoside, and (e) n-octyl-β-d-maltoside. Retention volumes of the major UV-absorbance peaks are labeled. 4

5 Supplementary Figure 3: Phasing and structure solution of ChbC. The final ChbC model is shown superposed with electron density calculated with (a) amplitudes and solvent-flattened experimental phases from the 4.5 Å Ta 6 Br 12 -derivatized dataset, contoured at 1.0 σ, (b) sigmaa-weighted 2F o -F c Fourier coefficients from the 3.3 Å native dataset and phases from a model containing only polyalanine, contoured at 1.0 σ, and (c) sigmaa-weighted 2F o -F c Fourier coefficients from the 3.3 Å dataset and phases from the final model, contoured at 1.5 σ. Panels on the right correspond to enlarged views of the area marked by a black rectangle on the left. 5

6 (% $%&!"-. %0& 34!!"#!"'!")!"*!",!"+!"-/ %0#. %0#/!"2!"#1!"& $%# Supplementary Figure 4: Membrane topology of ChbC. Topology diagram of ChbC with helices denoted as rectangles and ß-sheet as arrows. The diagram is oriented with the extracellular side on top. The helix coloring scheme is consistent with that used in Figure 1c and Supplementary Figure 1. The black lines show the approximate location of the membrane, and the N- and C-termini are marked with letters. 6

7 Supplementary Figure 5: Stereo-views of the ChbC dimer. Stereo-views of the ChbC dimer as viewed from (a) the intracellular side, (b) inside the plane of the membrane, and (c) the extracellular side. The two-fold rotational symmetry axis relating the protomers is marked in panel b. 7

8 Supplementary Figure 6: Crystal packing in the P Chbc crystals. ChbC dimers in a section of the crystal lattice are shown with the two protomers colored green and blue, and the ß-hairpins in both protomers colored purple. 8

9 Supplementary Figure 7: The ChbC dimer interface. The surfaces of the two protomers of ChbC are shown as opaque blue and transparent green viewed from within the plane of the membrane (left) and from the intracellular side (right): the residues involved in the dimer interface are colored black on the opaque subunit. 9

10 "#$! "#$!! "#( %&'! "#( %&$* %&+ %&) %&$* %&'- %&'- %&+ %&'! %&) %&, "#$- "#$- %&, - -((( +"*, %(." )"*$ -((( +"*, %(." )"*$ '((& %"&# '((& %"&# Supplementary Figure 8: The (GlcNAc) 2 binding site. (a) Stereo-view of the C- terminal domain and bound (GlcNAc) 2 molecule viewed from the plane of the membrane. The green mesh corresponds to F o -F c density calculated in the absence of (GlcNAc) 2 and contoured at 2.5 sigma. (b) Stereo-view of the sugar-binding pocket viewed from the intracellular side. A (GlcNAc) 2 molecule is shown modeled in the orientation placing the C6-OH of the non-reducing sugar (red arrow) closest to the cytoplasm, along with residues potentially forming hydrogen bonds or hydrophobic interactions with the sugar. 10

11 Supplementary Figure 9: Fit of diacetylchitobiose in the electron density. Stereoview of (GlcNAc) 2 placed in the ChbC sugar-binding site in two orientations: (a) with the non-reducing C6 -OH (red arrow) closer to the cytoplasm (used in the final model), and (b) with the non-reducing C6 -OH buried in the protein interior. The green mesh corresponds to the Fo-Fc difference electron density contoured at 3.0 σ. 11

12 Supplementary Figure 10: Speculative model of the ChbC transport mechanism. Possible conformational changes to convert the occluded, sugar-bound state observed in the crystal structure to the inward- and outward-facing open states are proposed. These motions could potentially be either concerted between the two protomers or could occur independently as depicted here. To convert the occluded state (middle) to the outward-facing state (left), an outward rigid body rotation of the region comprising TM8, HP1-2, and TM9-10 could expose the sugar-binding cavity to the periplasmic space. Opening of the intracellular gate to form the inward-open state (right) could be achieved by the straightening of helix TM5 on the neighboring protomer, as triggered by phosphorylation of the (GlcNAc)