ULTRA-HIGH RESOLUTION MACROMOLECULAR CRYSTALLOGRAPHY; SUBTILISIN AT 0.78 A. VISUALIZATION OF HYDROGEN

Richard Bott¹, Peter Kuhn², Mark Knapp³, Michael Soltis², Grant Ganshaw¹ and Michael Theone¹

¹ Genencor International, 925 Page Mill Road Palo Alto CA
² SSRL, 2575 Sand Hill Road, Menlo Park CA
³ LLNL, 7000 East Avenue, PO Box 808 Livermore CA

Crystals of subtilisin from Bacillus lentus when cryocooled, diffract to at least 0.75 A. Complete data has been collected to 0.78 A resolution. A total of 257583 unique reflections have been collected at SSRL beamline 9-1 with an overall R-merge of 4.1%. The coordinates have been refined using SHELXL-97 including aniostropic temperature factors to a R-factor of 10.9% when all data 20-0.78 A are included.

B. lentus subtilisin, a serine protease having MW=27,000, crystallizes in space group P2₁2₁2₁ with a=52.65, b=61.25 and c=74.75 A. The structure is essentially identical to a structure determined at room temperature to 1.8 A resolution. It is possible to differentiate C, N and O atoms and infer double and single bonds in favorable instances. We are presently including hydrogen atoms which are visible as 3s peaks in Fo-Fc difference electron density maps. We find one unusual hydrogen bond in the active site of the enzyme. This bond would correspond to an unusually downfield shifted proton reported for numerous serine proteases by NMR.