Protocol for purification, crystallization, and structure determination of the mpox virus mRNA cap N7 methyltransferase complex
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 E1<sub>CTD</sub>-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.<sup>1</sup>.
Authors
- Jixi Li (ORCID: https://orcid.org/0000-0003-3463-3175)
- Qingqing Guo (ORCID: https://orcid.org/0000-0003-2956-3491)
- Anke Chen
- Jin Ding (ORCID: https://orcid.org/0000-0003-3585-6063)
Institutions
- Fudan University (CN)
- Pudong Medical Center (CN)
Publication Details
- Journal
- STAR Protocols
- Published
- 2025-05-29
- DOI
- https://doi.org/10.1016/j.xpro.2025.103845
- Citations
- 1
- Primary Topic
- Poxvirus research and outbreaks
- Type
- article
- Field-Weighted Citation Impact
- 1.04
Funders
- National Natural Science Foundation of China