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[…] Recombinant SorT and SorU proteins were overproduced and purified as previously described (), with minor modifications. SorT was crystallized by hanging drop vapor diffusion with drops consisting of equal volumes (2 μL) of protein and crystallization solution (0.1 M HEPES pH 7.5, 8% ethylene glycol, 0.1 M manganese (II) chloride tetrahydrate and 17.5% PEG 10,000) at 20°C. Crystals were cryoprotected in reservoir solution with 30% glycerol before flash-cooling in liquid nitrogen. Small (ca. 20 × 10 × 10 μ) crystals of SorU were grown in drops containing equal volumes (2 μL) of protein and reservoir solution (1.8 M tri-sodium citrate, pH 5.5, 0.1 M glycine), which were harvested and flash-cooled in liquid nitrogen without additional cryoprotection. Purified SorT (20 mM Tris pH 7.8, 2.5% glycerol) and SorU (20 mM Tris pH 7.8, 150 mM NaCl) were mixed and incubated on ice at a molar ratio of 2:1 (SorU:SorT; total protein concentration 8 mgmL-1) before crystallization via hanging-drop vapor diffusion with a reservoir solution containing 0.2 M sodium formate, 0.1 M Bis-Tris propane pH 7.5 and 20% PEG 3350. Crystals grew to a maximum size of ca. 150 x 100 x 20 μ in 4 days at 20°C and were flash-cooled in liquid nitrogen after brief soaking in mother liquor containing 30% glycerol. All diffraction data were collected on an ADSC Quantum 315r detector at the Australian Synchrotron on beamline MX2 at 100 K and were processed with HKL2000 (). Unit cell parameters and data collection statistics are presented in .The crystal structure of SorT was solved by molecular replacement using PHASER () with a search model generated with CHAINSAW () from the SorA portion of the SorAB crystal structure (29.0% sequence identity, Protein Data Bank entry 2BLF []) as a template (). The resulting model was refined by iterative cycles of amplitude based twin refinement (using twin operators H, K, L and –H, -K, L with estimated twin fractions of 0.495 and 0.505 respectively) within REFMAC (), interspersed with manual inspection and correction against calculated electron density maps using COOT (). The refinement of the model converged with residuals R = 0.208 and Rfree = 0.239 (). The structure of the SorT/SorU complex was solved by molecular replacement using PHASER (), with the refined SorT structure as a search model. Initial rounds of refinement yielded a difference Fourier electron density map, which clearly showed positive difference density for the location of one molecule of SorU per asymmetric unit, which was manually built using COOT (). Refinement was carried out with REFMAC5 () and PHENIX () and converged with residuals R = 0.211 and Rfree = 0.260 (). The refined SorU model, from the SorT/SorU complex structure, was used as a search model to solve the SorU structure by molecular replacement using PHASER (). Refinement was carried out with REFMAC5 and PHENIX () and converged with residuals R= 0.192 and Rfree = 0.240. All structures were judged to have excellent geometry as determined by MOLPROBITY ()(). [...] SAXS analysis of the SorT/SorU complex was performed in a buffer of 20 mM Tris pH 7.8, 2.5% v/v glycerol. Purified SorU and SorT were mixed and incubated on ice at a molar ratio of 2:1 (SorU:SorT), generating two samples of total protein concentrations 2.75 and 6.25 mgmL-1, respectively. SAXS data were measured as described previously() with the data collection parameters listed in . Data were reduced to I(q) vs q (q=4πsinθλ,where q=4sin2θ is the scattering angle) using the program SAXSquant that includes corrections for sample absorbance, detector sensitivity, and the slit geometry of the instrument. Intensities were placed on an absolute scale using the known scattering from H2O. Protein scattering was obtained by subtraction of the scattering from the matched solvents (20 mM Tris pH 7.8, 2.5% v/v glycerol obtained from the flow-through after protein concentration by centrifugal ultrafiltration). Molecular weight (Mr) estimates for the proteins were made using the equation from Orthaber(): Mr=NAI(0)C△ρM2 where NAis Avogadro’s number, C is the protein concentration and △ρM=△ρυ, where △ρ is the protein contrast and υ the partial specific volume, both of which were determined using the program MULCh().The ATSAS program package() was used for data analysis and modeling, with the specific programs used detailed in , along with the data ranges and results of each of the calculations. Further detail on data interpretation and analysis for these experiments is detailed in Appendix 2. [...] Sulfite dehydrogenase enzyme assays were carried out as described previously (; ; ). The reduced – oxidized extinction coefficient for SorU at 550 nm was 17.486 mM-1 cm-1 as determined by spectroelectrochemistry. Data fitting was carried out using Sigmaplot 12 (Systat). […]

Pipeline specifications

Software tools Coot, REFMAC5, PHENIX, MolProbity, ATSAS, SigmaPlot
Applications Miscellaneous, Small-angle scattering, Protein structure analysis
Organisms Sinorhizobium meliloti, Bacteria
Chemicals Hydrogen