Reply to Graille, “InsPection of electron density maps supports wrongly modeled hexakisphosphate (InsP6) bound to African swine fever mRNA-decapping enzyme g5Rp”
Abstract
See affiliation list on p. 7.T he g5Rp protein in African swine fever virus (ASFV), a key member of the Nudix phosphohydrolase family, plays a critical role in mRNA degradation and the hydrolysis of diphosphoinositol polyphosphates.In our earlier report, we presented the crystal structures of g5Rp and its complex with inositol hexakisphosphate (InsP 6 ), elucidating the structural basis for InsP 6 's inhibitory effect on g5Rp-mediated mRNA decapping (1).This manuscript revisits our experimental evidence in response to critiques on the electron density map interpretation for InsP 6 , aiming to reinforce the accuracy and validity of our initial findings.Following the protocols detailed in our previous publication, we prepared both wild-type and selenomethionine-labeled g5Rp wild type proteins (Se-Met g5Rp).The complex was prepared by mixing the wild-type protein with InsP 6 in a 1:3 ratio.The crystallization of these proteins, achieved via sitting-drop vapor diffusion with commer cial screening kits.The resulting crystals of both g5Rp variants and the g5Rp-InsP 6 complex have been thoroughly documented and are available for review.All details regarding the buffers are comprehensively outlined in Table 1, covering those utilized in both protein purification and crystallization mother liquor.The process of protein purification is illustrated in Fig. 1.The exceptionally pure protein samples were uti lized to initiate the crystallization screening.Notably, the wild-type and…
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