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Fig. S1. A carbon monoxide difference spectrum of reduced CYP7B1.

Fig. S2. SDS/PAGE of CYP7B1 mutant forms.

Fig. S3. The HPLC chromatogram of a 25-hydroxycholesterol conversion by CYP7B1. The eluted fractions were further analyzed by LC-MS (inset).

Fig. S4. The HPLC chromatogram of a 5α-androstane-3-β-ol-17-one conversion by CYP7B1. The eluted fractions were further analyzed by LC-MS (inset).

Fig. S5. The multiple sequence alignment of CYP7A1 and CYP7B1 proteins from different organisms.

Fig. S6. NMR analysis of the major product of 25-hydroxycholesterol oxidation by CYP7B1.

Table S1. 1H NMR data for 25-hydroxycholesterol and the product of its conversion by CYP7B1 in CDCl3 (500 MHz).

Table S2. Steroids that showed low or no binding to CYP7B1.

Table S3. Azoles that showed low or no binding to CYP7B1.

Table S4. CYP7B1 mutations, their effects and structural role.

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