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, 116.2,, (º) 90,90,90 90,90,90 Peak Inflection Remote Wavelength (Å) 1.0750 0.9791 0.9793 0.9600 Resolution (Å) 50~3.6 (3.73~3.6) 50~3.8 (3.94~3.8) 50~3.8 (3.94~3.8) 50~3.8 (3.94~3.8) R merge(%) 0.065(0.831) 0.088 0.076 0.081 I/ I 32.2(2.2) 31.9(1.8) 29.8(1.4) 31.6(1.8) Completeness (%) 99.7(100) 99.8(100) 99.8(100) 99.8(100) Redundancy 6.9 10.3 10.3 10.3 Refinement Resolution (Å) 50~3.6 No. reflections 46,786 R work/ R free 0.265/0.289 No. atoms 2660 Protein 2606 Ligand 54 Water 0 B-factors(Å 2 ) Protein 142.6 Ligand 149.0 Water R.m.s deviations Bond lengths (Å) 0.009 Bond angles (º) 1.348 One crystal was used for each data set listed above. Highest resolution shell is shown in parenthesis. WWW NATURE.COM/NATURE 1
Supplementary Figure 1 RibU is the S component of the ECF transporter for riboflavin. a, Riboflavin is bound to RibU from Staphylococcus aureus (S. aureus). WWW NATURE.COM/NATURE 2
Shown here is an absorption spectrum of recombinant RibU protein, which indicates the presence of riboflavin. The wavelength scan of riboflavin alone (blue line) is shown as the control. In addition, mass spectrometric analysis of RibU confirmed the presence of riboflavin (data not shown). b, RibU formed a stable complex with the T, A, and A components of the putative ECF-type transporter for riboflavin. RibU and its corresponding T, A, and A proteins, all derived from S. aureus, were co-expressed in E. coli. The Gene ID and predicted molecular weight are: RibU: GI:161509653, 21.1 kda; A: GI:15925211, 32.9 kda; A : GI:15925212, 30.0 kda; T: GI:15925210, 30.8 kda. These four proteins formed a stable complex, which was co-purified by three sequential steps: affinity chromatography, anion exchange, and gel filtration. Shown here is the gel filtration chromatogram of the quaternary complex (indicated by the red line). The peak fractions from gel filtration were visualized on SDS-PAGE by coomassie staining. The protein bands corresponding to A, A, and T components were excised from the gel and subjected to complete proteolysis by trypsin. The trypsinized fragments were separated by HPLC and analyzed by mass spectroscopy. 36, 29, and 16 peptide fragments exactly matched the sequences of A, A, and T, respectively (data not shown). These results unambiguously confirmed the identity of the components. The A and A components could be co-expressed and co-purified by affinity chromatography and gel filtration (indicated by the blue line). See Method for details. c, The presence of all four components (RibU, T, A, and A, all from S. aureus) allowed the riboflavin-auxotrophic E. coli mutant strain BSV11 to grow in LB without added riboflavin. By contrast, the individual presence of RibU, T, RibU+T, or A+A, failed to support the growth of the mutant E. coli in LB without added riboflavin. An equal volume of the culture (10 μl) was dispensed onto the LB plates, occupying the upper half of each plate. The lower half of each plate was used WWW NATURE.COM/NATURE 3
to streak the culture from the upper half. The plates were incubated at 37 o C overnight. See the Method for details. Similar results were obtained for the E. coli mutant strain BSV13. BSV11 and BSV13 are E. coli mutant strains that have lost the ability to synthesize riboflavin de novo. WWW NATURE.COM/NATURE 4
Supplementary Figure 2 Electron density maps of RibU. a, A stereo view of the anomalous density map, contoured at 4.0, in each asymmetric unit. The four strongest peaks, each above 5.0, correspond to Met20 and Met123 in the two RibU molecules. The other two peaks shown, each above 3.0 _, correspond to Met9 and Met79 in one of the two RibU molecules. b, A stereo view of the 2Fo-Fc electron density map, contoured at 1.0, in each asymmetric unit. The protein main chain is colored yellow. c, A stereo view of the 2Fo-Fc electron density map, contoured at 1.0, around TM1 and TM2. d, A stereo view of the 2Fo-Fc electron density map, contoured at 1.0, around TM3 and TM4. e, A stereo view of the 2Fo-Fc electron density map, contoured at 1.0, around TM5 and TM6. f-g, Stereo views of the WWW NATURE.COM/NATURE 5
OMIT electron density map, contoured at 2.5, around the modeled riboflavin molecules in the two RibU molecules. WWW NATURE.COM/NATURE 6
Supplementary Figure 3 Structure of RibU in one asymmetric unit. a, Three mutually perpendicular views of the RibU homo-dimer in one asymmetric unit. The two molecules are colored blue and green, with their N- and C-termini labeled and the bound riboflavin molecules shown in yellow. b, Surface electrostatic potential of the RibU homo-dimer in one asymmetric unit. The left, middle, and right panels correspond to those in panel a. If the RibU dimer were biologically relevant, the membrane-spanning distance was only approximately 20 Å and patches of highly charged surface would be buried within the lipid membrane (middle panel). These features do not support the scenario that RibU may form a homo-dimer in lipid membrane as observed in the crystals. WWW NATURE.COM/NATURE 7
Supplementary Figure 4 Structure of a RibU molecule. a, Four mutually perpendicular views of the RibU molecule. The N- and C-termini are colored blue and red, respectively. The bound riboflavin molecule is shown in ball-and-stick. b, Surface electrostatic potential of the RibU molecule. The four views correspond to those in panel a. The outer surface of the RibU cylinder is predominantly hydrophobic, consistent with the notion that this surface may be in contact with the non-polar interior of the lipid bilayer. By contrast, both ends of the cylinder-shaped RibU are highly charged. WWW NATURE.COM/NATURE 8
Supplementary Figure 5 Sequence alignment of RibU from representative bacterial species. The amino acid sequences of RibU homologs from 8 bacterial species are aligned, with the secondary structural elements indicated above the sequences. Invariant residues are highlighted in red whereas conserved amino acids are boxed. Residues that may be hydrogen-bonded to riboflavin through side chain and main chain atoms are denoted by magenta and green triangles, respectively. Residue that may bind riboflavin through van der Waals interactions are identified by blue squares. Positively charged amino acids in L4 loop and the C-termini are shaded green. The 17 amino acids in the L1 loop are shaded blue. The RibU homologs are from Staphylococcus aureus (GI: 161509653 ), Lactococcus lactis (GI:30024032), Clostridium acetobutylicum (GI:15896096), Streptococcus pyogenes (GI: 15674521), Leuconostoc mesenteroides (GI:227431697), Enterococcus faecalis (GI:29376104), Pediococcus pentosaceus (GI:116493321), Symbiobacterium thermophilum (GI:51893863). WWW NATURE.COM/NATURE 9
Supplementary Figure 6 Structure of RibU is dissimilar to those of the transmembrane domains of the ABC transporters. Shown here is a structural comparison of RibU with three classes of ABC transporters. The N- and C-termini are indicated. Two perpendicular views of each structure are shown in the upper and bottom panels. WWW NATURE.COM/NATURE 10
Supplementary Figure 7 Structural comparison of RibU with hits of the DALI search. a, A stereo view of the structural overlay of RibU (blue) with particulate methane monooxygenase (pmmo) from Methylosinus trichosporium OB3B (colored orange, PDB accession code 3CHX). b, A stereo view of the structural overlay of RibU (blue) with pmmo from Methylococcus capsulatus (colored magenta, PDB accession code 1YEW). C, Structures of RibU and pmmo. The N- and C-termini are indicated. WWW NATURE.COM/NATURE 11
Supplementary Figure 8 Mapping of conserved amino acids onto the structure of RibU. Based on the sequence alignment of 12 RibU homologs, residues that are conserved in 7-9 and 10-11 bacterial species are colored yellow and orange, respectively. Invariant residues are highlighted in red. A ribbon diagram and a surface representation are shown. WWW NATURE.COM/NATURE 12
Supplementary Figure 9 Features of the riboflavin-binding pocket. a, A stereo view of the MAD experimental electron density map for riboflavin. The electron density map, colored magenta, is contoured at 0.7. The final model of riboflavin is shown here as a reference. b, Features of the riboflavin-binding pocket. All buried amino acids (left panels) are divided into two groups: non-polar (middle panels) and polar/charged (right panels). The polar/charged amino acids are predominantly located in a small region of RibU (identified by magenta circle in the right panel), around the L1 loop and TM3. By contrast, the hydrophobic residues are mainly located in TM4-6. Altogether, the L1 loop and the N-terminal portion of TM4 have a high density of polar and charged amino acids in the substrate-binding pocket, whereas the C- terminal portion of TM5 and the N-terminal portion of TM6 are predominantly WWW NATURE.COM/NATURE 13
hydrophobic. These structural features only support one way of orienting riboflavin into the binding pocket. WWW NATURE.COM/NATURE 14
Supplementary Figure 10 Riboflavin is recognized by conserved amino acids from L1 and TM 4-6. Detailed interactions are indicated in the two stereo panels. Residues in L1, TM4, TM5, and TM6 are shown in green, cyan, blue, and magenta, respectively. Hydrogen bonds are represented by red, dashed lines. WWW NATURE.COM/NATURE 15
Supplementary Figure 11 FMN, but not FAD, can be modeled into RibU. a, A slice of the riboflavin-bound RibU is shown for comparison. The surface of RibU is represented by blue mesh. The main chain of RibU is shown in blue ribbon. b, A close-up view of RibU with riboflavin replaced by FMN. The model was subjected to rigid body refinement. As can be seen, the extra phosphate group can be accommodated. c, FAD cannot be modeled into RibU. Modeling the adenine dinucleotide portion of FAD requires major structural rearrangements in RibU. WWW NATURE.COM/NATURE 16
Supplementary Figure 12 A working model of RibU. In this model, the L1 loop is predicted to regulate substrate binding: it remains open in the apo-transporter and closes down upon binding to substrate in the periplasm. TM1-3 are thought to move away from TM4-6, likely driven by ATP hydrolysis of the A component, and allows the substrate to be released into the cytoplasm. WWW NATURE.COM/NATURE 17
Supplementary Figure 13 The S components of ECF transporters are predicted WWW NATURE.COM/NATURE 18
to contain 6 TMs. The amino acid sequences of 17 ECF transporters are aligned with those of two RibU proteins from Staphylococcus aureus and Bacillus subtilis. Of the 17 transporters, 4 are specific for folate (FolT, vitamin B 9 ), 7 for thiamine precursor (HmpT, vitamin B 1 ), and 6 for cobalamin precursor (CblT, vitamin B 12 ). The corresponding secondary structural elements are shown for RibU. The conserved amino acids are highlighted in yellow, and the extent of sequence conservation is shown above the sequences by color-coded vertical bars. The two RibU proteins are from Staphylococcus aureus (RibU-Sa, GI: 161509653 ) and Bacillus subtilis (RibU- Bs, GI: 16079362). The 4 folate transporters are from Thermoanaerobacter tengcongensis (FolT-Tt, GI: 20808786), Enterococcus faecalis (FolT-Ef, GI: 238857495), Lactobacillus gasseri (FolT-Lg, GI: 116629049), and Pediococcus pentosaceus (FolT-Pp, GI: 116493189). The 7 thiamine transporters are from Thermoanaerobacter tengcongensis (HmpT-Tt, GI: 20807418), Alkaliphilus metalliredigens (HmpT-Am, GI: 150391423), Bacillus sp. B14905 (HmpT-Bb, GI: 126653120), Clostridium acetobutylicum (HmpT-Ca, GI: 15893922), Enterococcus faecalis (HmpT-Ef,GI: 29376490), Lactococcus lactis (HmpT-Ll, GI: 15672470), and Streptococcus pyogenes (HmpT-Sy, GI: 139473115). The 6 cobalamin precursor transporters are from Listeria monocytogenes (CblT-Lm, GI: 47095032), Alkaliphilus metalliredigens (CblT-Am, GI: 150387919), Bacillus sp. B14905 (CblT-Bb, GI: 126653029), Clostridium botulinum (CblT-Cb, GI: 153939773), Desulfitobacterium hafniense Y51 (CblT-Ds, GI: 89895470), and Geobacillus thermodenitrificans (CblT- Gt, GI: 138895830). WWW NATURE.COM/NATURE 19
Supplementary Figure 14 Alignment of ligand-specific ECF transporters reveals WWW NATURE.COM/NATURE 20
candidate sequences that are involved in ligand binding. a, Alignment of the S components of five folate transporters. The predicted secondary structural elements are shown above the sequences. The conserved amino acids are highlighted in yellow, and the extent of sequence conservation is indicated above the sequences by colorcoded vertical bars. The amino acid sequences that correspond to those of the riboflavin-binding site in RibU are indicated by thick magenta lines below the sequences. The five folate transporters are from Alkaliphilus metalliredigens (FolT- Am, GI: 150389000), Enterococcus faecalis (FolT-Ef, GI: 238857495), Lactobacillus gasseri (FolT-Lg, GI: 116629049), Pediococcus pentosaceus (FolT-Pp, GI: 116493189), and Thermoanaerobacter tengcongensis (FolT-Tt, GI: 20808786). b, Alignment of the S component of eight cobalamin precursor transporters. The eight cobalamin precursor transporters are from Alkaliphilus metalliredigens (CblT-Am, GI: 150387919), Bacillus sp. B14905 (CblT-Bb, GI: 126653029), Clostridium botulinum (CblT-Cb, GI: 153939773), Desulfitobacterium hafniense Y51 (CblT-Ds, GI: 89895470), Geobacillus thermodenitrificans (CblT-Gt, GI: 138895830), Listeria monocytogenes (CblT-Lm, GI: 47095032), Moorella thermoacetica (CblT-Mt, GI: 83589060), and Thermoanaerobacter tengcongensis (CblT-Tt, GI: 20806890). WWW NATURE.COM/NATURE 21
Supplementary Figure 15 The S components of the group II ECF transporters within the same bacterial species share some sequence features. Sequences of the predicted L4 loop and the C-terminus of the S components from Clostridium tetani and Streptococcus mutans are shown here. Clostridium tetani and Streptococcus mutans were chosen because, compared to other species, there are a large number of group II S components that share the same A-T module. The positively charged amino acids are colored green. WWW NATURE.COM/NATURE 22