Abstract
Atomic resolution structures (beyond 1.20 Å) at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes. Serial femtosecond crystallography (SFX) has attracted surging interest by providing a route to bypass such challenges. Yet the progress on atomic resolution analysis with SFX has been rather slow. In this report, we describe the 1.20 Å resolution structure of proteinase K using 13 keV photon energy. Hydrogen atoms, water molecules, and a number of alternative side-chain conformations have been resolved. The increase in the value of B-factor in SFX suggests that the residues and water molecules adjacent to active sites were flexible and exhibited dynamic motions at specific substrate-recognition sites.
Highlights
Atomic resolution structures at ambient temperature, which is usually hampered by the radiation damage in synchrotron X-ray crystallography (SRX), will add to our understanding of the structure-function relationships of enzymes
The disposition and the protonation state of hydrogen atoms and a number of water molecules can be resolved, which promotes our understanding of enzyme functions directly linked to their structure[6,7,8,9,10,11,12,13]
We report the 1.20 Å resolution structure of proteinase K obtained at ambient temperature
Summary
For SFX experiments, proteinase K from Engyodontium album (No P2308, Sigma) was crystalized by mixing a 1:1 ratio of 40 mg ml−1 protein solution in 20 mM MES–NaOH (pH 6.5) and precipitant solution composed of 0.5 M NaNO3, 0.1 M CaCl2, 0.1 M MES–NaOH (pH 6.5). A 1.0-ml sample of crystallization solution was centrifuged at 20 °C and 3,000 g for 3 min, and the supernatant solution was removed. Diffraction-quality crystals of proteinase K (size 100 × 100 × 200 μm) were obtained using the oil microbatch method[25]. X-ray diffraction images were collected using an RAXIS-V area detector (Rigaku, Tokyo, Japan) with synchrotron radiation in the wavelength of 0.80 Å at the BL26B1 station of SPring-8 (Hyogo, Japan)[38].
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