SUPPLEMENTARY INFORMATION

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Junior Hodges
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1 doi: /nature12045 Supplementary Table 1 Data collection and refinement statistics. Native Pt-SAD X-ray source SSRF BL17U SPring-8 BL41XU Wavelength (Å) Space group P P Unit cell (Å) a=79.97, b=148.69, c= a=80.6, b=150.56, c= Resolution (Å) 50~3.53 (3.66~3.53) 50~4.2 (4.35~4.2) R merge (%) 9.6 (88.8) 14.7 (87.6) I/σ I 13.9 (1.4) 12.1 (1.1) Completeness (%) 96.9 (97.9) 97.8 (88.6) Number of measured reflections 76, ,860 Number of unique reflections 22,601 14,477 Redundancy 3.4 (3.3) 10.8 (4.7) Wilson B factor (Å 2 ) FOM (Figure-Of-Merit) after DM R / R free (%) / Number of atoms / B-factor: All atoms 7436 / Main chain 3836 / Side chain 3566 / Detergent 34 / Ramachandran plot (%): Most favored 84.1 Additional allowed 12.7 Generously allowed 2.8 Disallowed 0.4 RMS-deviation: Bond distances (Å) Bond angles ( ) Values in parentheses are for the highest resolution shell. R free was calculated with 5% of the reflections selected randomly. 1

Supplementary Figure 1 Generation of a quaternary ECF transporter for crystallization. a, Gel filtration of a quaternary ECF transporter for crystallization.

2 Supplementary Figure 1 Generation of a quaternary ECF transporter for crystallization. a, Gel filtration of a quaternary ECF transporter for crystallization. The ECF transporter for hydroxymethyl pyrimidine 1 (Hmp) contains four components: two ATPases EcfA (residues 1-279) and EcfA (residues 1-290), a putative Hmp-binding component (residues 1-166), and an energy-coupling component EcfT (residues 1-266). The buffer for gel filtration (Superdex-200, GE Healthcare) contained 25 mm Tris-Cl, ph 8.0, 150 mm NaCl, and 0.2% (w/v) n-decyl-β-d-maltopyranoside (DM, Anatrace). b, Crystallization of the quaternary ECF transporter for Hmp. Two different crystal forms were obtained, one in a hexagonal space group (upper panel) and the other in P (lower panel). The hexagonal crystals diffracted X-rays weakly and were not pursued further. c, A representative X-ray diffraction image of the crystals in the P space group. 2

Supplementary Figure 2 Phase determination for the ECF transporter complex. a, A stereo view of the anomalous difference Fourier peaks for platinum atoms in each asymmetric unit.

3 Supplementary Figure 2 Phase determination for the ECF transporter complex. a, A stereo view of the anomalous difference Fourier peaks for platinum atoms in each asymmetric unit. The electron density is contoured at 5.0. A total of 10 platinum peaks, each higher than 5, were located in each asymmetric unit. All structural images in the Supplementary Information were prepared with PyMol 2. b, Analysis of the Figure-of-Merit (FOM) by resolution. The highest FOM right after PHASER was approximately 0.45 (red line, around 10 Å). After DM, the FOM was significantly improved to an overall value of for the resolution range of Å (blue line). 3

Supplementary Figure 3 Experimental electron density maps for the ECF complex. a, An overall view of the experimental electron density, contoured at 2.5 in one asymmetric unit.

4 Supplementary Figure 3 Experimental electron density maps for the ECF complex. a, An overall view of the experimental electron density, contoured at 2.5 in one asymmetric unit. b, Four representative, close-up views of the experimental electron density map, contoured at 1.5, are shown around key regions of the EcfT and EcfS components. The top two panels show the experimental electron density around the two coupling helices of EcfT: 6 (top left) and 7 (top right). The bottom two panels display the experimental electron density around two helices of EcfS that contribute to the bulk of interactions with EcfT: 1 (bottom left) and 6 (bottom right). 4

Supplementary Figure 4 Representative 2Fo-Fc electron density map for the ECF complex. a, An overall view of the 2Fo-Fc electron density, contoured at 2.5 in one asymmetric unit.

5 Supplementary Figure 4 Representative 2Fo-Fc electron density map for the ECF complex. a, An overall view of the 2Fo-Fc electron density, contoured at 2.5 in one asymmetric unit. b, Four representative, close-up views of the 2Fo-Fc electron density map, contoured at 1.5, are shown for key regions of the EcfT and EcfS components. The top two panels show the 2Fo-Fc electron density around the two coupling helices of EcfT: 6 (top left) and 7 (top right). The bottom two panels display the 2Fo-Fc electron density around two helices of EcfS that contribute to the bulk of interactions with EcfT: 6 (bottom left) and 1 (bottom right). 5

Supplementary Figure 5 Sequence alignment of EcfT and its functional homologues from six representative bacterial species. The secondary structural elements are shown above the sequences.

Residues that interact with EcfA/A through van der Waals contacts are indicated by magenta squares.

6 Supplementary Figure 5 Sequence alignment of EcfT and its functional homologues from six representative bacterial species. The secondary structural elements are shown above the sequences. Invariant amino acids among all six proteins are shaded in red whereas conserved amino acids are colored red. Residues that interact with EcfA/A through van der Waals contacts are indicated by magenta squares. Residues that may use their side chains or main chains to form hydrogen bonds (H-bonds) with EcfA/A are identified by red and blue triangles, respectively. Residues that interact with EcfS are identified by purple asterisks. The EcfT homologues are from Lactobacillus brevis (GI: ), Bacillus subtilis (GI: ), Thermoanaerobacter tengcongensis (GI: ), Staphylococcus aureus (GI: ), Lactococcus lactis (GI: ), Streptococcus mutans (GI: ). 6

Supplementary Figure 6 Topology and coupling elements of the energy coupling module EcfT. a, Topology diagram of EcfT. EcfT exhibits a previously unreported protein fold, which we term the ECF fold.

7 Supplementary Figure 6 Topology and coupling elements of the energy coupling module EcfT. a, Topology diagram of EcfT. EcfT exhibits a previously unreported protein fold, which we term the ECF fold. The C-terminal halves of helices 6 and 7 serve as the main coupling module for EcfA and EcfA, respectively. b, Conserved organization between the ATPase domain and its coupling module. EcfA (magenta) and its coupling module in EcfT (green) are superimposed with EcfA (yellow) and its coupling module in EcfT (cyan). The C-terminal portions of 6 and 7 adopt nearly identical positions with respect to the ATPases. 7

8 Supplementary Figure 7 Sequence alignment of L. brevis EcfA and EcfA with functional orthologs from five additional bacterial species. The corresponding 8

9 secondary structural elements in Lactobacillus brevis EcfA and EcfA are indicated above the sequences. The conserved ABC transporter motifs are labeled under the sequences. The 5 sets of EcfA and EcfA proteins are from Lactobacillus brevis (GI: and ), Bacillus subtilis (GI: and ), Staphylococcus aureus (GI: and ), Lactococcus lactis (GI: and ), and Streptococcus mutans (GI: and ). Two NBDs of ABC transporters, MalK (GI: , PDB code 3FH6 3, chain A) and BtuD (GI: , PDB code 2QI9 4, chain C), are all from Escherichia coli for this alignment. 9

Supplementary Figure 8 Structural features of the ATPases EcfA and EcfA. a, Structure of EcfA/EcfA contains three subdomains: RecA-like, helical, and C-terminal subdomains.

10 Supplementary Figure 8 Structural features of the ATPases EcfA and EcfA. a, Structure of EcfA/EcfA contains three subdomains: RecA-like, helical, and C-terminal subdomains. Shown here in the left panel is EcfA. Arrangement of these three subdomains is similar to that of the NBD MalK (PDB code 3RLF 5, shown on the right panel). b, Key residues for ATP binding and hydrolysis are highly conserved in EcfA/EcfA. Shown here in the left panel is structural superposition between EcfA (rainbow color) and MalK (grey). A close-up view of the ATP-binding site is shown on the right panel. The ATP analogue AMPPNP (stick representation) and the magnesium ion (red sphere) are from MalK 5. Some of the key residues from EcfA are shown: Lys46 and Ser47 in the P-loop or Walker A motif, Gln88 in the Q-loop, Asp165 in the Walker B motif, and His199 in the H-loop. All these amino acids are invariant between EcfA/EcfA and MalK. 10

Supplementary Figure 9 The two ATPases EcfA and EcfA exist in an open conformation in the absence of nucleotide binding. The EcfA-EcfA hetero-dimer is shown in two perpendicular views.

11 Supplementary Figure 9 The two ATPases EcfA and EcfA exist in an open conformation in the absence of nucleotide binding. The EcfA-EcfA hetero-dimer is shown in two perpendicular views. In the left two panels, the EcfA-EcfA hetero-dimer is viewed on top of the RecA-like and helical subdomains. In the right two panels, the EcfA-EcfA hetero-dimer is viewed laterally along the RecA-like and C-terminal subdomains of EcfA and the helical subdomain of EcfA. In the absence of nucleotide binding, there is a gap between the two ATPases, and homo-dimerization is mediated mainly by the C-terminal domains, particularly by the two -helices at the C-terminus (top right panel). 11

Supplementary Figure 10 Sequence alignment of the putative Hmp-binding component EcfS from L. brevis with representative homologues from other bacterial species.

12 Supplementary Figure 10 Sequence alignment of the putative Hmp-binding component EcfS from L. brevis with representative homologues from other bacterial species. The corresponding secondary structural elements in the putative Hmp-binding component EcfS from Lactobacillus brevis are indicated above the sequences. The putative substrate-binding pocket of EcfS contains six polar or charged amino acids: Glu41, His84, Gln87, Tyr120, Asn 140, and Gln143. These six residues, identified by green triangles under the sequences, are either invariant or highly conserved among the EcfS homologues shown here. These residues are proposed to be involved in biding to the substrate molecule. The 6 putative Hmp-binding EcfS proteins are from Lactobacillus brevis (GI: ), Enterococcus faecalis (GI: ), Streptococcus pyogenes (GI: ), Lactococcus lactis (GI: ), Thermoanaerobacter tengcongensis (GI: ), and Alkaliphilus metalliredigens (GI: ). 12

Supplementary Figure 11 The interface between the substrate-binding component EcfS and the energy-coupling component EcfT. a, Three representations of the EcfS-EcfT subcomplex.

The middle panel shows EcfS in cartoon and EcfT in electrostatic potential. b, A close-up view of the interface between EcfS and EcfT.

13 Supplementary Figure 11 The interface between the substrate-binding component EcfS and the energy-coupling component EcfT. a, Three representations of the EcfS-EcfT subcomplex. The left panel shows both components in cartoon, whereas the right panel displays both components by surface electrostatic potential. The middle panel shows EcfS in cartoon and EcfT in electrostatic potential. b, A close-up view of the interface between EcfS and EcfT. The left panel shows the interface between 1 of EcfS and surrounding structural elements of EcfT. The right panel displays the interface between 6 of EcfS and surrounding structural elements of EcfT. All interacting residues are shown in stick. To avoid confusion, only amino acids from EcfS are labeled. 13

The functional motion of the entire ECF transporter system was computed using the coarse-grained anisotropic network model, where all four components EcfT/EcfS/EcfA/EcfA are treated as an elastic

14 Supplementary Figure 12 Functional motion simulation of the ECF transporter suggests direction of potential protein movement. a, Substrate entry point of EcfS is open to the cytoplasm in this snapshot. The functional motion of the entire ECF transporter system was computed using the coarse-grained anisotropic network model, where all four components EcfT/EcfS/EcfA/EcfA are treated as an elastic network and every residue is treated as one bead with a spring connecting each pair of beads positioned within a distance limit of 11 Å. The effect of the membrane bilayer on the system is also taken into account implicitly. The first functional motion mode obtained from this computation was chosen and shown in the Supplementary Movie generated using VMD. Shown here in panels a and b are two snapshots of EcfS relative to EcfT. It should be noted that such analysis suggests the direction in which protein will distort, not how far it will go in that direction. For clarity of viewing, EcfA and EcfA are omitted from these two panels. b, Substrate entry point of EcfS is open to the periplasm in this snapshot. c, Overlay of the EcfS-EcfT subcomplex shown in panels a and b. 14

15 References 1. Rodionov, D.A. et al. A novel class of modular transporters for vitamins in prokaryotes. J Bacteriol 191, (2009). 2. DeLano, W.L. The PyMOL Molecular Graphics System. on World Wide Web (2002). 3. Khare, D., Oldham, M.L., Orelle, C., Davidson, A.L. & Chen, J. Alternating access in maltose transporter mediated by rigid-body rotations. Mol Cell 33, (2009). 4. Hvorup, R.N. et al. Asymmetry in the structure of the ABC transporter-binding protein complex BtuCD-BtuF. Science 317, (2007). 5. Oldham, M.L. & Chen, J. Snapshots of the maltose transporter during ATP hydrolysis. Proc Natl Acad Sci U S A 108, (2011). 15

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Data collection Supplementary Table 1 Statistics of data collection, phasing and refinement Native Se-MAD Space group P2 1 2 1 2 1 P2 1 2 1 2 1 Cell dimensions a, b, c (Å) 50.4, 94.2, 115.4 49.8, 94.2,