Additional file 4: Figure S3. of Crystal structure of human S100A8 in complex with zinc and calcium

Calibration of the Size Exclusion Chromatography (SEC) column and composition of the two oligomeric forms obtained by SEC for hS100A8. (a) Theoretical molecular weights and measured elution volumes of the different protein standards used for calibration of the 24 ml SEC column (Superdex 75, GE Healthcare Lifesciences). The calibration was performed in 20 mM HEPES pH 7.5, 200 mM NaCl and 5 mM CaCl2. (b) Calibration curve derived from the SEC runs with the different protein standards. (c) Elution profile of hS100A8 on the SEC column equilibrated in 20 mM HEPES pH 7.5, 200 mM NaCl and 5 mM CaCl2. In the sole presence of calcium, hS100A8 elutes as two peaks with elution volumes of 11.15 (Peak 1) and 12.80 (Peak 2), respectively. (d) Molecular weights for the two hS100A8 species and corresponding molecular composition extrapolated from the calibration curve. The equation used to calculate the molecular weights is indicated above the table. (PDF 107 kb

Additional file 1: Table S1. of Crystal structure of human S100A8 in complex with zinc and calcium

Data collection and processing statistics for the datasets collected at wavelengths of 1.27 Å and 1.30 Å for both crystal forms 1 and 2. Values for the outer shell are given in parentheses. All datasets were processed with XDS [45] with the Friedel pairs kept separated. (DOCX 24 kb

Additional file 2: Figure S1. of Crystal structure of human S100A8 in complex with zinc and calcium

Identification of the new Zn-sites in the Zn2+/Ca2+-hS100A8 structure derived from crystal form 2 (C2221). (a) Experimental map containing anomalous data obtained after SAD-phasing in PHENIX.AUTOSOLVE [37] displayed as magenta mesh and contoured at 3.5 σ. The final refined model is superimposed for comparison. 12 anomalous sites were identified, including the 8 Zn2+ sites and 4 of the 8 Ca2+ sites (Ca2, Ca3, Ca6 and Ca8). (b) Anomalous difference Fourier map calculated using phases and weight from the best refined atomic model (without ions) obtained with the native dataset (crystal form 2) and anomalous differences from the datasets collected at a wavelength of 1.27 Å (magenta mesh, contour at 3.5 σ). (c) Anomalous difference Fourier map calculated using phases and weight from the best refined atomic model (without ions) obtained with the native dataset (crystal form 2) and anomalous differences from the datasets collected at a wavelength of 1.30 Å (red mesh, contour at 3.5 σ). The anomalous signal disappears for all Zn2+ ions but remains for the Ca2+ ions. (PDF 1749 kb

Structures of Alkaloid Biosynthetic Glucosidases Decode Substrate Specificity

Two similar enzymes with different biosynthetic function in one species have evolved to catalyze two distinct reactions. X-ray structures of both enzymes help reveal their most important differences. The <i>Rauvolfia</i> alkaloid biosynthetic network harbors two <i>O</i>-glucosidases: raucaffricine glucosidase (RG), which hydrolyses raucaffricine to an intermediate downstream in the ajmaline pathway, and strictosidine glucosidase (SG), which operates upstream. RG converts strictosidine, the substrate of SG, but SG does not accept raucaffricine. Now elucidation of crystal structures of RG, inactive RG-E186Q mutant, and its complexes with ligands dihydro-raucaffricine and secologanin reveals that it is the “wider gate” of RG that allows strictosidine to enter the catalytic site, whereas the “slot-like” entrance of SG prohibits access by raucaffricine. Trp392 in RG and Trp388 in SG control the gate shape and acceptance of substrates. Ser390 directs the conformation of Trp392. 3D structures, supported by site-directed mutations and kinetic data of RG and SG, provide a structural and catalytic explanation of substrate specificity and deeper insights into <i>O</i>-glucosidase chemistry