Nature Protocols: doi: /nprot Supplementary Figure 1

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1 Supplementary Figure 1 Influence of ZnCl 2 concentration on ph of 50 (gray) and 100 mm (orange) Tris-HCl buffer solutions. The green line represents the initial value of the ph of Tris buffers.

2 Supplementary Figure 2 Fluorescence spectra and fluorescence scans for ESA complexed with four different metals. Data for ESA complexed with copper (A), gold (B), mercury (C), and platinum (D) are shown. The figure represents screenshots generated by the data collection program JBluIce-EPICS as implemented on APS GM/CA-CAT beamlines. For each panel, the top screenshot shows the fluorescence emission spectrum collected with the excitation energy on or slightly above the theoretical value of the metal absorption edge ( ev). This spectrum displays the characteristic emission peak for the metal of interest (bracketed by the red boundaries) as well as the incident beam peak. Note that there are no significant peaks for other metals in these spectra. The middle screenshot shows the fluorescence emission spectrum collected with the excitation energy at ev below the metal absorption edge; the characteristic peak for the metal of interest is absent on this spectrum. The bottom screenshot shows the fluorescence absorption scan collected with the excitation energy in the range ±30 ev of the tabulated metal absorption edge; note that the emitted fluorescence is measured, which is proportional to the absorbed energy. The energy of the absorption edge, approximated as the inflection f point (indicated by the orange vertical line and listed in the table below each graph as infl ), is close to the table values for each metal. Note that the width of the absorption edges (the energy difference between the absorbance inflection point and its peak; these values coincide with f inflection point and its peak) for both Cu K edge and Hg L-III edge is much wider than those for Au L-III and Pt L-III. In the case of Hg L-III edge, the range of the excitation energy could be increased since the typical range of ±30 ev does not fully cover the absorption edge. The optimal energy for collecting X-ray diffraction data above the absorption edge is the maximum of f (i.e. the maximum of absorption and fluorescence), which is located at the top of the fluorescence scan and indicated by the green vertical line. The optimal energy for collecting X-ray diffraction data below the absorption edge is the highest energy below the absorption edge that gives only background fluorescence signal (virtually flat area of f ).

3 Supplementary Table 1. Examples of differences between the ph of the stock buffer solution and the final ph of crystallization cocktail. Data were collected from the C6 Webtool at CSIRO Collaborative Crystallization Centre 1. Screen name Well Conditions Buffer ph Final ph C3_1 - Peggy: Low MW, diverse buffers, ph range and salts. A1 10% v/v jeffamine M-600, ph=7.0; 0.1 M trisodium citrate-citric acid, ph=5.5; 0.01 M iron(iii) chloride 7.0 and C3_1 - Peggy: Low MW, diverse buffers, ph range and salts. C3_1 - Peggy: Low MW, diverse buffers, ph range and salts. C3_7 - Organics: MPD, 'small' branched polymers, hexanediol. C3_7 - Organics: MPD, 'small' branched polymers, hexanediol. Salty: Ammonium sulfate, lithium sulfate & trisodium citrate. A2 G7 20% v/v jeffamine M-600, ph=7.0; 0.05 M magnesium chloride; 0.05 M potassium chloride 33% w/v polyethylene glycol 600; 0.2 M DLmalate-imidazole, ph=5.5 F8 20% w/v pentaerythritol ethoxylate (15/4 EO/OH); 0.1 M tris chloride, ph=8.5 G8 B2 30% w/v pentaerythritol propoxylate (17/8 PO/OH); 0.1 M potassium thiocyanate 1.3 M ammonium sulfate; 0.1 M sodium MES, ph=6.5; 0.2 M ammonium dihydrogen phosphate No buffer

4 Supplementary Table 2. Percentage of protein-mn, -Fe, -Ni, -Cu, and -Zn complexes that were deposited into the PDB and collected at the wavelength corresponding to the appropriate metal absorption K-edge (here, the range of 100 ev below and 50 ev above the metal absorption edge was considered and is referred to as correct wavelengths). Wavelength (Å) Metal ID in PDB Metal No. of all structures with metal No. of structures collected on correct wavelength* Percentage of structures collected on correct wavelength* MN3 Mn MN Mn FE2 Fe FE Fe NI Ni CU1 Cu CU Cu ZN Zn * It is possible that the depositors chose to report only one dataset even if they collected multiple datasets since only one structure factor file is required for PDB deposition. Therefore, these statistics may not fully reflect the data collection practice used by scientists. The one dataset reported with its corresponding wavelength is usually the one with highest resolution (often the commonly used selenium K-edge). This dataset is not necessarily the one collected at a wavelength at the metal absorption edge. Even if datasets above and below the metal absorption edge were collected for a particular structure, they could be of lower resolution (than the one deposited into the PDB). The lower resolution datasets may be only used to identify the location of the metal but not deposited into the PDB. 2

5 Supplementary Table 3. Determination of metal concentration in a protein sample after elution from a nickel affinity column by ICP-OES. The analyzed sample is a molybdenum cofactor-containing chaperone protein (UniProt ID: H9NN97) involved in maturation of a molybdoenzyme-steroid C25 dehydrogenase. * - below detection limit Element Concentration [mg/l] Lower detection limit [mg/l] Ag ± Al* ± As not detectable 0.1 B* ± Ba* ± Be not detectable Bi* ± Ca* ± Cd* ± Co not detectable 0.01 Cr* ± Cs not detectable Cu ± Fe ± K 0.41 ± Li* ± Mg not detectable 0.1 Mn not detectable Mo* ± Na 444 ± Ni ± P* 0.3 ± Pb not detectable 0.01 S 5.1 ± Si* 0.06 ± Sr* ± Te not detectable 0.2 Ti* ± Tl not detectable 0.2 V not detectable 0.05 Zn 0.02 ±

6 Supplementary Table 4. Crystallization and cryoprotection conditions for the ESA-Zn 2+ and HSA-Zn 2+ complexes. (Adapted from Handing et al 2 ). PDB ID Zn 2+ conc. (mm) ph Albumin Final conc. (mm) 5IJF HSA 0.7 Crystallization conditions 0.1 M MMT (DL-Malic acid, MES monohydrate, Tris) Buffer ph 9.0, 23% w/v PEG 1500, 1 mm ZnCl2 5IIH IIU IIX ESA M Li2SO4 0.1 M Tris:HCl ph 7.4 / 6.9 / 6.5 / M (NH4)2SO4 5 mm ZnCl2 Soaking No No Yes Yes Yes No 5IJE IJ M (NH4)2SO4 0.1 M Na acetate 0.1 M ZnCl2 ZnCl 2 Final conc. in the crystallization drop (mm) Additional cryoprotectant None Paratone-N 50% Paratone-N, 50% Mineral Oil 4

7 REFERENCES 1. Newman, J., Fazio, V. J., Lawson, B. & Peat, T. S. The C6 Web Tool: A Resource for the Rational Selection of Crystallization Conditions. Cryst. Growth Des. 10, (2010). 2. Handing, K. B. et al. Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins. Chem. Sci. 7, (2016). 5