Crystallization, data collection and structure determination

The purified hNaa60(1-242), hNaa60(1-199) or F34A(1-199) protein was mixed with acetyl coenzyme A (Ac-CoA) or coenzyme A (CoA) (Sigma), respectively, at a 1:5 molar ratio before crystallization. All crystals were made by the hanging-drop vapor diffusion method. The crystallization reservoir solution for hNaa60(1-242) was 10 mM Tris pH 8.0, 75 mM NaCl, 0.5% glycerol, 3% v/v Tacsimate pH 4.0 (Hampton Research) and 7.5% w/v polyethylene glycol 3350 (PEG 3350), and for hNaa60(1-199) was 0.2 M L-Proline, 0.1 M HEPES pH 7.5, 10% w/v PEG 3350. Crystals of F34A mutation were obtained in 0.2 M Lithium Sulfate monohydrate, 0.1 M Tris pH 8.5, 20% w/v PEG 3350. The crystals were flash-frozen in liquid nitrogen in a cryo-protectant made of the reservoir solution supplemented with 25% glycerol.

The diffraction data were collected at the Shanghai SSRF BL18U1 beamline or at the Argonne National Laboratory APS ID19 beamline at 100 K. The data were processed with HKL3000³⁰. The hNaa60(1-199) structure was determined by molecular replacement with Phaser³¹ using a previously reported GNAT family acetyltransferase structure (PDB 2AE6) as the search model. The hNaa60(1-242) structure was solved by molecular replacement using hNaa60(1-199) structure as the search model. To improve the model quality, the programs ARP/wARP³² in CCP4 or simulated-annealing in CNS³³ were used. Iterative cycles of manual refitting and crystallographic refinement were performed using COOT³⁴ and Phenix³⁵. Ac-CoA/CoA and malonate were modeled into the closely fitting positive Fo-Fc electron density and then included in following refinement cycles. Topology and parameter files for Ac-CoA/CoA and malonate were generated using PRODRG³⁶. All figures for the molecular models were prepared using the PyMOL program. Statistics of diffraction data processing and structure refinement are shown in Table 1.