The high-resolution X-ray crystallography of bovine H-protein of glycine cleavage system

4 4 catalyze hydrolysis of the glycosidic linkages in the polygalacturonic acid of pectin. The reaction undergoes the general acid base catalysis in which proton transfer plays the most important role of the mechanism. Thus, we determined the sub-atomic-level structure of endoPG I from Stereum purpureum by joint X-ray and neutron diffraction. The enzyme was expressed in E. coli Origami and crystallized by a macro seeding method. For the X-ray study, the crystals (ca 0.25 mm 3 ) were soaked into a cryoprotectant solution and flash frozen in liquid nitrogen. X-ray diffraction data (10 -0.62 ) was collected at beam line BL41XU in SPring-8 using a helium cryostat (40 K). Rmerge, Rp.i.m.. and completeness of the X-ray data are 6.3% (50.0% in the most outer shell), 2.4% (21.6%) and 93.7% (55.0%), respectively. The final model of X-ray (R = 8.8%, Rfree = 9.8%) contains 2,433 hydrogen. For the neutron study, the crystals (ca 4.0 mm 3 ) were soaked into a D2O solution to decrease incoherent scattering from hydrogen. Neutron diffraction data (20 -1.5 ) was collected using BIX-4 in JRR-3 reactor of JAEA at room temperature. Rmerge and completeness of the neutron data are 11.7% (24.8%) and 89.1% (68.5%), respectively. The final model of neutron (R = 21.3%, Rfree = 23.0%) contains 2,227 hydrogen and 301deuterium. Visibility of hydrogen and deuterium atoms on the electron and nuclear density map will be presented.