Fig 2: (a) Active-site structure of the 2-deoxy-2-fluoro-ß-d-glucopyranoside-GBA covalent intermediate (PDB entry 6tjq). The 2F-Glc moiety is covalently bound to the catalytic nucleophile (Glu340), which occupies two conformations. a/b, catalytic acid/base; Nuc, catalytic nucleophile; EDO, ethylene glycol. Electron density is contoured to 1.1s (0.40 e Å-3). (c) Mechanism of the hydrolysis of 2F-DNPGlc by GBA to generate the covalent glycosyl-enzyme intermediate

Fig 3: (a) Electron density for active-site residues, including the catalytic nucleophile (Nuc) Glu340 and catalytic acid/base (a/b) Glu235, contoured to 2s (1.0 e Å-3). (b) Selection of modelled residues with difference electron density [green; contoured to 3s (0.37 e Å-3)] highlighting proton positions. (c)–(f) Modelled residues from domain I of GBA (PDB entry 6tn1) demonstrating atomic resolution (electron density contoured to 3.5s (1.75 e Å-3).

Fig 4: (a) Optimization of the crystallization pH using bis-Tris propane buffer. (b) Optimization of the protein concentration. (c) Crystal structure of the GBA dimer obtained at 1.56 Å resolution (PDB entry 6tjk). N-Glycans are depicted in glycoblock format (McNicholas & Agirre, 2017 ?). (d) GBA monomer comprising of three domains: domain I (residues 1–27 and 383–414) is shown in blue, domain II (residues 30–75 and 431–497) in red and domain III (residues 76–381 and 416–430) in gold. The active site contains bound bis-Tris propane, which forms hydrogen bonds to Trp179, Asn234, Glu235, Glu340, Trp381 and an ethylene glycol (EDO) cryoprotectant molecule. Electron density is contoured to 1s (0.34 e Å-3). (e) Overlay of recombinant GBA (gold) obtained at pH 7.0 and Cerezyme (teal) obtained at pH 4.6 (PDB entry 6tjj).

Fig 5: Purification of recombinant GBA from cell-culture medium with purification chromatograms and SDS–PAGE analyses for each purification step. GBA (~55 kDa) was extracted from the medium by hydrophobic interaction chromatography followed by two rounds of cation-exchange chromatography with the addition of Tween 80 detergent. Purification was completed by a size-exclusion step to remove Tween 80.