Supplemental Data SUPPLEMENTAL FIGURES

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1 Supplemental Data CRYSTAL STRUCTURE OF THE MG.ADP-INHIBITED STATE OF THE YEAST F 1 C 10 ATP SYNTHASE Alain Dautant*, Jean Velours and Marie-France Giraud* From Université Bordeaux 2, CNRS; Institut de Biochimie et Génétique Cellulaires, 1 rue Camille Saint-Saëns, Bordeaux cedex, France. *Corresponding authors: Alain Dautant and Marie-France Giraud; Phone: Fax: ; A.Dautant@ibgc.cnrs.fr and Marie-France.Giraud@ibgc.cnrs.fr SUPPLEMENTAL FIGURES Fig S1. Silver-stained SDS-PAGE analysis of the purified F 1 F 0 -ATP synthase. Lane 1: fraction loaded onto a Superdex 200 gel filtration column (16 μg of enzyme). Lane 2: eluted fraction of the major peak (5 μg of enzyme). 1

2 Fig S2. Schematic slab view of the yf 1 c 10 :ADP crystal packing orthogonal to the b axis and along the [ 1 10 ] direction. The correspondence between the functional subunit name (α E, α TP, α DP, β DP, β E, β TP, γ, δ and ε) and the chain label (A to H) can be found on the same color patterns. The rotation direction of the ATP synthesis is indicated. When the molecule is oriented upwards, the central stalk is visible and (α/β) 3 is colored in orange. When oriented downwards, the central stalk is hidden and (α/β) 3 is colored in blue. The α DP - and β TP -subunits of one molecule are in contact with the α E - and β DP -subunits of the neighboring molecule. The α TP - and β E -subunits are widely solvent accessible so TP closure cannot be attributed to intermolecular contacts. 2

3 Fig S3. Protein sequence alignment of c-subunits (B. taurus, S. cerevisiae, I. tartaricus, P. modestum, S. oleracea, S. platensis and E. coli). To get a sequence alignment including more species (Na + or H + bacterial chloroplast and mitochondrial), see (50). The GxGxGx(G/A) motif is colored in yellow, the essential E/D in magenta, the R(N/Q)P motif in red and orange background. The preferred stoichiometry of the c-ring is indicated. Green stars denote residues involved in Na + binding of I. tartaricus Na + F-ATPase (16). 3

4 Fig S4. The 2F o -F c electron density map contoured 1.3 σ of yf 1 c 10 :ADP around the β TP catalytic site. For the sake of clarity, the ADP molecule and some of the side chains involved in nucleotide binding are only shown in the catalytic site. The backbones of the α TP - and β TP -subunits are colored in orange and green, respectively. The Mg 2+ ion is shown as a sphere colored in cyan. 4

5 Fig S5. Comparison of α TP -subunits of yf 1 c 10 :ADP and yf 1 (I) (14). The α TP -subunit can be split into two sub-domains I (residues α TP and α TP ) and II (residues α TP and α TP ). The sub-domains I and II are superimposed on the left and right parts of the figure, respectively. The whole yf 1 c 10 :ADP subunit is colored in blue whereas the sub-domains I and II of yf 1 (I) are colored in green and red, respectively. 5

6 Fig S6. Four serial cross-sections perpendicular to the pseudo 3-fold axes ( ) are viewed from the top of F 1. The three α-subunits are superimposed. For the sake of clarity, the (αβ) 3 catalytic heads of yf 1 c 10 :ADP (multicolored) and yf 1 (I) (green) are only shown). The nucleotides of yf 1 c 10 :ADP are drawn in ball-and-stick and Mg 2+ as a sphere. The central stalks are drawn as cartoon except for the coiled-coil of γ- subunits (γ1-50 and γ ), which are drawn as linked Cα. They are colored in blue for bf 1 :DCCD (13), green for yf 1 (I) (14) and red for yf 1 c 10 :ADP. The yf 1 c 10 :ADP ring (multicolored surface) and the rotor axis (yellow decagon) are drawn. (A) The C-terminal helices γ are stick in the proline-rich collar (αpro 290, αpro 291, βpro 276 ). The conserved γgly 273(268) is near the α DP - and β DP -subunits. (B) At the end of the coiled-coil γ1-17 and γ , the Tyr 255(250) backbone is more twisted in yeast. (C) Near the hinge residues, γ18-25 and γ , the yeast and bovine N-terminal helices clearly deviate. The arrow marks the deviation of 3 Å of the tip α TP toward the β TP -subunit. (D) The foot of the yeast yf 1 (I) central stalk is rotated 36 relative to the bovine stalk in the direction of ATP hydrolysis. 6

7 SUPPLEMENTAL TABLES Table S1: Buried surface area (Å 2 ) of interfaces between α- and β-subunits in F 1 -ATPase structures. α-β interfaces α E β E (E) β E α TP α TP -β TP (TP) β TP α DP α DP -β DP (DP) β DP -α E yf 1 c 10 :ADP wpd Pdb Id yf 1 (I) hld (13) yf 1 (III) hld (13) yf 1 (II) hld (13) yf 1 apo (I) fks (14) yf 1 apo (III) fks (14) yf 1 apo (II) fks (14) bf 1 :AMP-PNP bmf (3) bf 1 :DCCD e79 (12) bf 1 :ADP w0k (10) Table S2: Buried surface area (Å 2 ) of interfaces involving central stalk subunits in F 1 -ATPase structures. * ε-subunit is incomplete, ** δ- and ε-subunits are highly incomplete or absent. γ interfaces γ α E γ β Ε γ α TP γ β TP γ α DP γ β DP γ δ γ ε δ ε Ref yf 1 c 10 :ADP wpd yf 1 (I)* (13) yf 1 (II)** (13) bf 1 :AMP-PNP** (3) bf 1 :DCCD (12) bf 1 :ADP** (10) Table S3: Buried surface area (Å 2 ) of F 1 -F 0 rotor interfaces in yf 1 c 10 :ADP. Chain name J K L M N O P Q R S c 10 δ γ ε

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1

Nature Structural & Molecular Biology: doi: /nsmb Supplementary Figure 1 Supplementary Figure 1 Identification of the ScDcp2 minimal region interacting with both ScDcp1 and the ScEdc3 LSm domain. Pull-down experiment of untagged ScEdc3 LSm with various ScDcp1-Dcp2-His 6 fragments.