Human α-thrombin was purchased from Haematologic Technologies (catalog no., HCT-0020). The protein-stabilizing agent was removed by using PD-10 desalting column (GE Healthcare) equilibrated with 20 mM tris-HCl and 200 mM NaCl (pH 8.0). Buffer-exchanged human α-thrombin was incubated with the macrocycle P2 at a molar ratio of 1:3 and subsequently concentrated to 7.5 mg/ml by using a 3000 MWCO Vivaspin ultrafiltration device (Sartorius Stedim Biotech GmbH). Further P2 macrocycle was added during the concentration procedure to ensure that the inhibitor was present at a molar excess of at least threefold.

Crystallization trials of the complex were carried out at 293 K in a 96-well two-drop MRC (Medical Research Council) crystallization plate (Hampton Research, CA, USA) using the sitting-drop vapor diffusion method and the JCSG-plus, PACT premiere, Morpheus, and LMB crystallization screens (Molecular Dimensions Ltd., Suffolk, UK). Droplets of 600-nl volume (with a 1:1 protein:precipitant ratio) were set up using an Oryx 8 crystallization robot (Douglas Instruments Ltd., Berkshire, UK) and equilibrated against 80 μl of reservoir solution. Crystals appeared within 2 to 3 days in two different conditions: (i) 100 mM sodium citrate and 20% (w/v) polyethylene glycol, molecular weight 3000 (PEG 3000) (pH 5.5) and (ii) 100 mM MES and 15% (w/v) PEG 3350 (pH 6.2). Further attempts to optimize the conditions were performed by varying the protein complex concentration, the drop volume, and the protein:precipitant ratio. Best crystals were obtained using 100 mM MES and 15% (w/v) PEG 3350 (pH 6.2) as precipitant agent. For x-ray data collection, crystals were mounted on CryoLoops (Hampton Research, CA, USA), soaked in cryoprotectant solution [25% ethylene glycol, 100 mM MES, and 15% (w/v) PEG 3350 (pH 6.2)], and flash cooled in liquid nitrogen.

X-ray diffraction data of human α-thrombin in complex with P2 were collected at the ID29 beamline of the European Synchrotron Radiation Facility (Grenoble, France). The best crystals diffracted to a maximum solution of 2.30 Å. Crystals belong to the P212121 space group with unit cell dimensions a = 55.94 Å, b = 80.91 Å, and c = 159.43 Å. The asymmetric unit contains two molecules, corresponding to a Matthews coefficient of 2.67 Å3/Da and a solvent content of about 54% of the crystal volume. Frames were indexed and integrated with software XIA2 via the automatic processing protocol available at the beamline, merged, and scaled with AIMLESS (CCP4i2 crystallographic package).

The structure was solved by molecular replacement with the software PHASER, and the model 3U69 was used as a template. Refinement was carried out using REFMAC and Phenix. Rebuilding and fitting of the macrocycle were performed manually with graphic software COOT. Since the first cycles of refinement, the electron density corresponding to a portion of the bound peptide was visible in the electron density map. The refined model contains 4975 protein atoms, 141 P2 ligand atoms, 150 water molecules, and 48 atoms of other molecules. The final crystallographic R factor is 0.20 (Rfree, 0.236). Geometrical parameters of the model are as expected or better for this resolution. The solvent-excluded volume and the corresponding buried surface were calculated by the 3V web server using a spherical probe of 1.5 Å radius. Intramolecular and intermolecular hydrogen bond interactions were analyzed by ProFunc, LigPlot+, and PyMOL software.

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