Sequential removal of Ca 2+ from satellite tobacco necrosis virus : Crystal structure of two EDTA-treated forms

Abstract

Two forms of EDTA-treated satellite tobacco necrosis virus (STNV) have been studied with X-ray crystallography methods. The crystals of both forms were isomorphous with native STNV crystals, and ( F EDTA- F nat) maps as well as (2 F EDTA- F nat) maps were calculated with phases from the native structure. The maps were based on partial data sets to 2.8 Å resolution, and averaged using the 60-fold non-crystallographic symmetry. In the first crystal form, calcium ions were absent from one of the three sites in the icosahedral protein shell. The crystals were produced at pH 5.0 from a virus solution treated with EDTA at pH 6.5. The virions were not expanded, and no essential changes were seen in the protein shell. In the second crystal form, all calcium ions in the protein shell were absent. The virus material in these crystals had been subjected to treatment with EDTA at pH 8.0 before crystallization at pH 6.5. The high pH treatment caused degradation of the viral RNA. No expansion of the virion had occurred and all protein-protein contacts were retained. These results are compared with the previously presented low-resolution structure of slightly expanded STNV with intact RNA, where calcium ions from two sites were absent. The relevance of Ca 2+-depleted virions for infection in vivo is discussed as well as the possibility that the Ca 2+-binding sites may be parts of ion channels in the viral capsid. One possible RNA-binding site was found in the maps of both crystal types, and the same site could also be localized in the high-resolution map of native STNV.