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Multi-target Parallel Processing Approach for Gene-to-structure Determination of the Influenza Polymerase PB2 Subunit
Published on: June 28, 2013
Protocol for purification, crystallization, and structure determination of the mpox virus mRNA cap N7
Qingqing Guo1, Anke Chen1, Jin Ding2
1School of Life Sciences and Pudong Hospital, Fudan University, Shanghai Engineering Research Center of Industrial Microorganisms, Shanghai 200438, China.
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The mpox virus (MPXV) mRNA cap N7 methyltransferase (RNMT) methylates guanosine at mRNA 5'-cap N7 positions to enable immune evasion. Here, we present a protocol for E1CTD-E12 complex purification and crystallization. We describe steps for rational sequence design of the complex, co-expression in E. coli, affinity chromatography purification, gel filtration, and crystallization optimization using vapor diffusion. We further outline X-ray diffraction data collection and structure determination. This reproducible framework enables structural analysis of viral mRNA-modifying enzyme complexes. For complete details on the use and execution of this protocol, please refer to Chen et al.1.
