Structure of a serine protease proteinase K from Tritirachium album limberat 0.98 angstrom resolution

X-ray diffraction data at atomic resolution to 0.98 Angstrom with 136 380 o bserved unique reflections were collected using a high quality proteinase K crystals grown under microgravity conditions and cryocooled. The structure has been refined anisotropically with REFMAC and SHELX-97 with R-factors o f 11.4 and 12.8%, and R-free-factors of 12.4 and 13.5%, respectively. The r efined model coordinates have an overall rms shifts of 0.23 Angstrom relati ve to the same structure determined at room temperature at 1.5 Angstrom res olution. Several regions of the main chain and the side chains, which were not observed earlier have been seen more clearly. For example, amino acid 2 07, which was reported earlier as Ser has been clearly identified as Asp. F urthermore, side-chain disorders of 8 of 279 residues in the polypeptide ha ve been identified. Hydrogen atoms appear as significant peaks in the F-o - F-c difference electron density map accounting for an estimated 46% of all hydrogen atoms at 2 sigma level. Furthermore, the carbon, nitrogen, and ox ygen atoms can be differentiated clearly in the electron density maps. Hydr ogen bonds are clearly identified in the serine protease catalytic triad (S er-His-Asp). Furthermore, electron density is observed for an unusual, shor t hydrogen bond between aspartic acid and histidine in the catalytic triad. The short hydrogen bond, designated "catalytic hydrogen bond", occurs as p art of an elaborate hydrogen bond network, involving Asp of the catalytic t riad. Though unusual, these features seem to be conserved in other serine p roteases. Finally there are clear electron density peaks for the hydrogen a toms associated with the O gamma of Ser 224 and N delta1 of His 69.