Abstract
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.