Protein Expression and Purification—A truncated extracellular form of human ACE2 (residues 1-740) was expressed in baculovirus and purified as described previously (8 (link)). The signal sequence (residues 1-18) is presumably removed upon secretion from Sf9 cells. The molecular mass of the purified enzyme is 89.6 kDa by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, which is greater than the theoretical molecular mass of 83.5 kDa expected from the sequence (residues 19-740). The difference of ∼6 kDa is believed to be due to glycosylation at the seven predicted N-linked glycosylation sites for this protein.
Crystallization—Briefly, 2 μl of purified ACE2 (5 mg/ml) was combined with an equal volume of reservoir solution, and crystals were grown by hanging drop vapor diffusion at 16-18 °C. The best crystallization reservoir solution conditions for native ACE2 were found to be 100 mm Tris-HCl (pH 8.5), 200 mm MgCl2, and 14% polyethylene glycol 8000. Under these conditions, it took ∼2 weeks to grow single crystals suitable for x-ray diffraction. Similarly, diffraction-quality ACE2 crystals were also grown in the presence of an ACE2 inhibitor, MLN-4760 (ML00106791; (S,S)-2-{1-carboxy-2-[3-(3,5-dichlorobenzyl)-3H-imidazol-4-yl]-ethylamino}-4-methylpentanoic acid). Compound MLN-4760 corresponds to compound 16 of Dales et al. (15 (link)). Crystallization trials used 2 μl of reservoir solution plus 2 μl of ACE2 at 5.9 mg/ml containing 0.1 mm inhibitor. The best diffracting ACE2-inhibitor complex crystals were grown in the presence of 19% polyethylene glycol 3000, 100 mm Tris-HCl (pH 7.5), and 600 mm NaCl.
Data Collection and Structure Determination—The best data set for native ACE2 was at 2.2-Å resolution and was collected at the Advanced Photon Source (Argonne National Laboratory). A total of 44 x-ray data sets were collected for native ACE2, including a large number of heavy atom soaks of atoms that had good anomalous signals. The data sets for each derivative were collected at different wavelengths to maximize the anomalous signals for the bound heavy atoms. Native ACE2 data were collected to 2.2-Å resolution at λ = 1.28 Å to maximize the anomalous signal at the zinc absorption edge.
The heavy atom positions were determined and confirmed by a combination of visual inspection of Patterson maps and automatic search procedures, which included SHAKE 'N BAKE (16 (link)) and SHELXD (17 (link)). The heavy atom parameters were refined and optimized using the computer programs SHARP (18 (link)), MLPHARE (19 ), and XHEAVY (20 ). The experimental phases were improved by solvent flattening and histogram matching.
Once the native ACE2 structure was determined, it was used to solve the inhibitor-bound structure of ACE2 to 3.0-Å resolution using molecular replacement methods that employed the program AMoRe in the CCP4 software suite (21 (link)). The native structure was split into two subdomains: subdomains I and II (see Fig. 3for definition). Subdomain II was used for molecular replacement and refined in REFMAC5, which resulted in the appearance of electron density for subdomain I. Subdomain I was then fitted into the density by hand, and the structure was refined as a whole. Final refinement was accomplished using the software suite CNX (22 (link)).

Overview of the native ACE2 crystal structure.A, α-carbon trace of the native ACE2 structure looking down into the metallopeptidase active site cleft. The metallopeptidase catalytic domain is colored red. The active site zinc ion is shown as a yellow sphere, and the single bound chloride ion is shown as a green sphere. The S1′ subsite for inhibitor and substrate binding is to the right of the zinc ion, and the S1 subsite is to the left. The collectrin homology domain at the C terminus is disordered and denoted by the green dotted line. B, ribbon diagram of native ACE2 showing the secondary structure and also the two subdomains (I and II) that form the two sides of the active site cleft. The two subdomains are defined as follows: the N terminus- and zinc-containing subdomain I (red), composed of residues 19-102, 290-397, and 417-430; and the C terminus-containing subdomain II (blue), composed of residues 103-289, 398-416, and 431-615. This definition is based on motion observed upon inhibitor binding (see Fig. 4). Zinc and chloride ions are denoted as described for A.