Supplementary Figure 1

Influence of ZnCl2 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.

Nature Protocols: doi:10.1038/nprot.2018.018

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 (+10-20 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 30-50 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”).

Nature Protocols: doi:10.1038/nprot.2018.018

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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 Centre1.

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 5.5 5.7

C3_1 - Peggy: Low

MW, diverse buffers,

pH range and salts.

A2 20% v/v jeffamine M-600, pH=7.0; 0.05 M

magnesium chloride; 0.05 M potassium chloride

7.0 7.7

C3_1 - Peggy: Low

MW, diverse buffers,

pH range and salts.

G7 33% w/v polyethylene glycol 600; 0.2 M DL-

malate-imidazole, pH=5.5

5.5 6.5

C3_7 - Organics: MPD,

'small' branched

polymers, hexanediol.

F8 20% w/v pentaerythritol ethoxylate (15/4

EO/OH); 0.1 M tris chloride, pH=8.5

8.5 8.1

C3_7 - Organics: MPD,

'small' branched

polymers, hexanediol.

G8 30% w/v pentaerythritol propoxylate (17/8 PO/OH);

0.1 M potassium thiocyanate

No buffer 6.3

Salty: Ammonium

sulfate, lithium sulfate

& trisodium citrate.

B2 1.3 M ammonium sulfate; 0.1 M sodium MES,

pH=6.5; 0.2 M ammonium dihydrogen phosphate

6.5 5.8

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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*

1.8961 MN3 Mn3+ 20 0 0.00

1.8961 MN Mn2+ 2479 21 0.85

1.7433 FE2 Fe2+ 581 28 4.82

1.7433 FE Fe3+ 1277 20 1.57

1.4879 NI Ni2+ 983 27 2.75

1.3808 CU1 Cu+ 177 15 8.47

1.3808 CU Cu2+ 1035 40 3.86

1.2837 ZN Zn2+ 10878 419 3.85

* 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.

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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.

Element Concentration [mg/L] Lower detection limit [mg/L]

Ag 0.0716 ± 0.0006 0.01

Al* 0.007 ± 0.002 0.01

As not detectable 0.1

B* 0.043 ± 0.003 0.1

Ba* 0.0010 ± 0.0005 0.01

Be not detectable 0.005

Bi* 0.002 ± 0.009 0.01

Ca* 0.002 ± 0.004 10

Cd* 0.002 ± 0.0007 0.01

Co not detectable 0.01

Cr* 0.0010 ± 0.0008 0.01

Cs not detectable 0.005

Cu 0.006 ± 0.002 0.005

Fe 0.071 ± 0.003 0.01

K 0.41 ± 0.03 0.2

Li* 0.0002 ± 0.00005 0.005

Mg not detectable 0.1

Mn not detectable 0.005

Mo* 0.110 ± 0.002 0.2

Na 444 ± 5 0.1

Ni 0.110 ± 0.003 0.005

P* 0.3 ± 0.2 0.5

Pb not detectable 0.01

S 5.1 ± 0.1 1

Si* 0.06 ± 0.02 0.1

Sr* 0.00004 ± 0.00004 0.2

Te not detectable 0.2

Ti* 0.0006 ± 0.0002 0.02

Tl not detectable 0.2

V not detectable 0.05

Zn 0.02 ± 0.01 0.01

* - below detection limit

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Supplementary Table 4. Crystallization and cryoprotection conditions for the ESA-Zn2+ and HSA-Zn2+ complexes.

(Adapted from Handing et al2).

PDB ID

Zn2+ conc. (mM)

pH

5IJF

0.5

9.0

5IIH

2.5

7.4

5IIU

10

6.9

5IIX

15

6.5

5IJE

30

7.4

5IJ5

50

4.5

Albumin

Final conc. (mM)

HSA

0.7

ESA

0.2

Crystallization conditions 0.1 M MMT (DL-Malic acid,

MES monohydrate, Tris)

Buffer pH 9.0,

23% w/v PEG 1500,

1 mM ZnCl2

0.2 M Li2SO4

0.1 M Tris:HCl pH 7.4 / 6.9 / 6.5 /7.4

2.0 M (NH4)2SO4

5 mM ZnCl2

2.0 M (NH4)2SO4

0.1 M Na acetate

0.1 M ZnCl2

Soaking No No Yes Yes Yes No

ZnCl2

Final conc. in the

crystallization drop (mM)

0.5 2.5 10 15 30 50

Additional cryoprotectant None Paratone-N 50% Paratone-N,

50% Mineral Oil

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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, 2785–2792 (2010).

2. Handing, K. B. et al. Circulatory zinc transport is controlled by distinct interdomain sites on mammalian

albumins. Chem. Sci. 7, 6635–6648 (2016).

Nature Protocols: doi:10.1038/nprot.2018.018