研究目的
To understand the detailed mechanism of hydroxymethylation catalyzed by dCMP hydroxymethylase from bacteriophage T4 (T4dCH) by determining its high-resolution structure using X-ray free electron laser (XFEL) technique.
研究成果
The study successfully determined the high-resolution structure of T4dCH D179N mutant using XFEL, revealing a dTMP molecule at the active site that mimics the methylene intermediate. This provides insights into the hydroxymethylation mechanism, particularly the role of a critical water molecule in the final hydroxylation step.
研究不足
The study is limited by the resolution of the XFEL structure and the interpretation of the electron density map for the ligand. The mechanism proposed is based on the structure of a mutant enzyme, which may not fully represent the wild-type enzyme's behavior.
1：Experimental Design and Method Selection:
The study utilized the XFEL technique to determine the high-resolution structure of T4dCH D179N mutant at room temperature.
2：Sample Selection and Data Sources:
Microcrystals of T4dCH D179N mutant were prepared and used for data collection.
3：List of Experimental Equipment and Materials:
XFEL facility, microcrystals, monoolein mixture, LCP injector, and mass spectrometry equipment.
4：Experimental Procedures and Operational Workflow:
Microcrystals were mixed with a viscous medium and injected using an LCP injector. Diffraction data were collected and processed to determine the structure.
5：Data Analysis Methods:
The structure was determined by molecular replacement and refined using PHENIX and COOT. Mass spectrometry was used for ligand identification.
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