Crystallization, data collection, and structural determination

Plasma-derived hAPC-Gla-domainless (APC-GD) was purchased from Enzyme Research Laboratories and characterized by ELISA to confirm that it could be recognized by both C25K23Fab and hu1573Fab. For APC-C25K23Fab complex formation, 0.9 mg of APC was mixed with 1.05 mg of C25K23Fab, and the reaction mixture was incubated at 4 °C for 5 h. The mixture was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with HEPES-buffered saline (HBS, 10 mM HEPES, 150 mM NaCl, pH 7.2) 3 times to separate free APC or free Fab from the APC-C25K23Fab complex. Each fraction was collected and analyzed by SDS-PAGE under nonreducing and reducing conditions. The fractions containing the APC-C25K23Fab complex were pooled and concentrated to 16 mg/mL. For APC–hu1573Fab complex formation, 0.25 mg of APC was mixed with 0.25 mg of hu1573Fab, and the reaction mixture was incubated overnight at 4 °C. The mixture was loaded onto a Superdex 200 10/300 GL column (GE Healthcare) equilibrated with HBS buffer (10 mM HEPES, 150 mM NaCl, pH 7.2) to separate free APC or free Fab from the APC–hu1573Fab complex. Each fraction was collected and analyzed by SDS-PAGE under nonreducing and reducing conditions. This process was repeated 2–4 times, and the fractions containing the APC–hu1573Fab complex were pooled and concentrated to 15 mg/mL.

Crystallization of APC–C25K23Fab complex or APC–hu1573Fab complex under different crystal growth conditions was performed to produce crystals suitable for structure determination. The crystallization instrument Gryphon (Art Robbins Instruments, Sunnyvale, CA, USA) and ten commercial high-throughput crystallization screening kits (Hampton Research, Aliso Viejo, CA, USA, and Molecular Dimensions, Altamonte Springs, FL, USA) were used, and several hits were identified. The 2 hits for APC-C25K23Fab complex were as follows: (1) 0.1% n-octyl-beta-d-glucoside, 0.1 M sodium citrate tribasic dehydrate pH 5.5, 22% (w/v) PEG 3350, and (2) 18% 2-propanol, 0.1 M sodium citrate tribasic dehydrate, pH 5.5, 20% (w/v) PEG 4000. The best hits for aPC–hu1573Fab complex were (1) 0.2 M sodium fluoride, 20% (w/v) PEG 3350, (2) 0.1 M MES, pH 6.0, 0.2 M zinc acetate, 10% (w/v) PEG 8000, and (3) 0.1 M cacodylate, pH 6.5, 0.2 M lithium sulfate, 30% (v/v) PEG 400.

Diffraction data of crystals for both complexes were collected at the BL17U beamline at the Shanghai Synchrotron Research Facility. Diffraction data were indexed, integrated, and scaled with the program HKL2000⁴² (HKL Research, Inc., Charlottesville, VA, USA). The structure was determined by molecular replacement with PHASER⁴² in the CCP4 suite⁴³ using the published APC (PDB code: 1AUT)¹⁶ and Fab crystal structures as models. Iterative refinement of individual coordinates was performed with COOT⁴⁴ and PHENIX⁴⁵, and model adjustment yielded models with R_work of 0.201 and R_free of 0.241 at a resolution of 2.2 Å for APC–C25K23Fab complex and R_work of 0.278 and R_free of 0.302 at a resolution of 3.7 Å for APC–hu1573Fab complex. All structural figures were made with PyMOL (PyMOL Molecular Graphics System, Schrödinger, LLC, Mannheim, Germany). Data processing and refinement statistics are listed in Table 1.