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					Supplementary Information for

Structural insight into G protein coupling and regulation of Fe2+ membrane transport
Amy Guilfoyle1, Megan Maher1,6, Mikaela Rapp1,5,6, Ronald Clarke2, Stephen Harrop3, and Mika Jormakka1,4,*

1

Structural Biology Program, Centenary Institute, Locked Bag 6, Sydney, New South Wales 2042,

Australia.
2

School of Chemistry, University of Sydney, Sydney, NSW 2006, Australia Department of Biophysics, University of New South Wales, Sydney, New South Wales 2052, Australia. Faculty of Medicine, Central Clinical School, University of Sydney, Sydney, New South Wales 2006,

3

4

Australia.
5

Current Address: Division of Biophysics, Department of Medical Biochemistry and Biophysics,

Karolinska Institute, SE-171 77 Stockholm, Sweden
6

These authors contributed equaly Correspondence: m.jormakka@centenary.org.au (M.J.)

*

Supplementary Methods
Expression and Purification of FeoB1-280 The sequence corresponding to residues 1-280 of FeoB was sub-cloned from a full-length FeoB-GFP construct and inserted into a pGEX4-T1 vector (GE Healthcare Life Sciences). The sequenced expression vector was transformed into BL21 cells, and protein expressed as a fusion protein with Glutathion S-transferase (GST). Large scale cultures were initiated by inoculating 2 x 25 mL cultures from a glycerol stock of pGEX-FeoB1-280 BL21. The overnight cultures were subsequently used to inoculate 4 x 500mL cultures supplemented with 100g/mL Ampicillin. Cultures were incubated at 37 C until OD600 reached 0.6, where after the temperature were reduced to 30C. After the cultures had equilibrated, 1mM of IPTG were added. Cultures were incubated for a further 4 hours, before harvested by centrifugation. Cell pellets were re-suspended in ~100 mL 20 mM Tris pH 8.0 + 100 mM NaCl (Buffer A), and stored at -20C.

For protein purification, cells were thawed at room temperature before cell lysis by 3 passes in a continuous flow Avesting C3 homogenizer. The slurry was subsequently centrifuged at ~70,000 x g to remove un-broken cells and cell debris. Supernatant was thereafter incubated with GST affinity resin, pre-equilibrated with Buffer A, for one hour at 4C. Un-bound protein were eluted using a polyprep column (BioRad), and the resin was subsequently washed with 30 x column volumes of Buffer A. The GST moiety was removed by adding 50 units of thrombin to the resin, resuspended in 2 column volumes Buffer A + 10 mM MgCl2 + 10 mM CaCl2, and incubated for 48 hours at 30C. Cleaved protein was eluted and concentrated to ~2mL before loading on a Superdex 75 size exclusion chromatography column (GE Healthcare Life Sciences), pre-equilibrated with Buffer A. Protein eluted in a single peak at a molecular weight corresponding to monomeric FeoB1-280. Purified protein was buffer exchanged to 20 mM Tris pH 8.0 and concentrated to 20 mg ml-1 and stored at 4C. Structure Determination and Refinement of apo and liganded FeoB1-180

The structure of FeoB1-280 was determined by SIRAS phasing, using data from two single crystals: (1) a Pb derivative crystal, soaked in trimethyllead acetate (resolution limit 2.9 Å) and (2) a native crystal (resolution limit 2.2 Å; isomorphous with the Pb derivative data; Rmerge (Pb derivative versus native) = 0.26; Table S1). The positions of 6 Pb atoms per asymmetric unit were determined with SHELXD using the SAD protocol. These sites were used to calculate initial SIRAS phases, using both Pb derivative and native data sets. The phases were calculated to the resolution limit of the Pb derivative data (2.9 Å) using the program SHARP (Bricogne et al, 2003). The NCS matrices relating three FeoB1-280 molecules per asymmetric unit were calculated using the coordinates of the Pb heavy atom positions using PHENIX (Zwart et al, 2008). Density modification was carried out to the resolution limit of the native data (2.2 Å) using the program DM with solvent fattening, histogram mapping and averaging options, resulting in an interpretable experimental electron density map. The atomic model was built using the programs O (Jones et al, 1991) and COOT (Emsley & Cowtan, 2004). During the model building process, it was rapidly recognized that significant regions of the FeoB1-280 structure were disordered in the native crystals. The coordinates of the preliminary model were therefore used as a molecular replacement model to determine the structure of FeoB1-280 in crystals grown from the Se-Met labeled protein (space group C2), using PHASER (McCoy et al, 2007). Model building was completed using this data and refinement was carried out using REFMAC5 (Murshudov et al, 1997) (with TLS) and PHENIX (Zwart et al, 2008). Anisotropy-corrected data provided by the diffraction anisotropy server was used for density modification and refinement (Strong et al, 2006). Since the data from the Se-Met labeled protein produced the most complete model, this structure will hereafter be referred to as apo-FeoB1-280. The coordinates of the apo-FeoB1-280 model (monomer chain A) were used as a molecular replacement model to find the positions and orientations of three FeoB1-280 molecules per asymmetric unit in the mGTP-FeoB1-280 (space group P21, 2.74 Å) data set using PHASER. Refinement of the mGTP-FeoB1-280 structure was carried out with REFMAC5 (with TLS) and PHENIX. For both refinements, water molecules were added

automatically using ARP/wARP

(Perrakis et al, 1999). Tight NCS restraints were

maintained throughout the refinement of the apo-FeoB1-280 structure. No NCS restraints were applied for the refinement of the mGTP-FeoB1-280 structure.

Quality and Description of the Structures The structure of FeoB1-280 was solved by SIRAS phasing using data from trimethyl lead derivative and native crystals. The initial model was built using the native data in space group P212121. However, it was rapidly evident that this structure suffered from

significant regions of disorder, which was presumably due to a lack of crystal packing interactions between molecules in the crystal. Therefore, the incomplete model, built in space group P212121 was used as a molecular replacement model to solve the structures of a mant-GTP-FeoB1-280 complex (mGTP-FeoB1-280, space group P21) and a Se-Met labeled derivative of FeoB1-280 (apo-FeoB1-280, space group C2). Although the two structures belong to different space groups, they both have a common composition, with three FeoB1-280 molecules (related by a three-fold non-crystallographic symmetry axis) per asymmetric unit. Since strict non-crystallographic symmetry restraints were retained throughout the refinement of the apo-FeoB1-280 structure, all structural descriptions are for molecule A only. The refinement of both FeoB1-280 structures converged with residuals less than 23%. They show excellent geometry with 100.0% of residues located in the allowed region of a Ramachandran plot (Davis et al, 2007). Both structures show some degree of disorder, with no ordered electron density being observed for varying regions of each structure. Accordingly, these regions (corresponding to approximately 9 and 13% of the sequence of the apo-FeoB1-280 and mGTP-FeoB1-280 structures, respectively) were omitted from the final models (Table 1).

The presence of disorder in these structures can be simply explained by the packing of molecules in the crystals. For example, the mGTP-FeoB1-280 structure has three main regions of disorder: residues 31-39, residues 65-70 and residues 262-280. These regions of the structure are not found in the vicinity of intermolecular contacts, either between

molecules in the trimer or between trimers. In contrast, residues 31-39 are well ordered in all three chains of the apo-FeoB1-280 structure. These regions of the apo-FeoB1-280 monomers participate in interactions with other crystallographic symmetry-related molecules in the crystal (for example, ValA30 makes a main chain hydrogen bond with ThrB*37 of a symmetry-related molecule and ThrA37 interacts with ValC*30). The monomer structures of mGTP-FeoB1-280 and apo-FeoB1-280 can be superposed with an rmsd in C positions of 1.03 Å (245 common C positions). This value reflects the change in conformation observed for the G5 loop. In fact, the maximum distances for C positions after superposition of the structure are for residues Thr151 and Arg152 (8.1 and 6.1 Å, respectively).

Supplementary References
Bricogne G, Vonrhein C, Flensburg C, Schiltz M, Paciorek W (2003) Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0. Acta Crystallogr D Biol Crystallogr 59: 20232030. Davis IW, Leaver-Fay A, Chen VB, Block JN, Kapral GJ, Wang X, Murray LW, Arendall WB, Snoeyink J, Richardson JS, Richardson DC (2007) MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res 35: W375-W383. Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60: 2126-2132. Jones TA, Zou JY, Cowan SW, Kjeldgaard M (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr A 47 ( Pt 2): 110-119. McCoy AJ, Grosse-Kunstleve RW, Adams PD, Winn MD, Storoni LC, Read RJ (2007) Phaser crystallographic software. 40: 658-674. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240-255. Perrakis A, Morris R, Lamzin VS (1999) Automated protein model building combined with iterative structure refinement. Nat Struct Biol 6: 458-463. Strong M, Sawaya MR, Wang S, Phillips M, Cascio D, Eisenberg D (2006) Toward the structural genomics of complexes: crystal structure of a PE/PPE protein complex from Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 103: 8060-8065. Zwart PH, Afonine PV, Grosse-Kunstleve RW, Hung LW, Ioerger TR, McCoy AJ, McKee E, Moriarty NW, Read RJ, Sacchettini JC, Sauter NK, Storoni LC, Terwilliger TC, Adams PD (2008) Automated structure solution with the PHENIX suite. Methods Mol Biol 426: 419-435.


				
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