Journal: Nature Communications
Article Title: Iron-sulfur clusters in SARS-CoV-2 exoribonuclease and methyltransferase complexes: relevance for viral genome proofreading and capping
doi: 10.1038/s41467-025-62832-5
Figure Lengend Snippet: a UV-visible absorption spectra of nsp10 and nsp14, individually or co-expressed and purified from Expi293F cells, showing peaks at ~420 nm characteristic of [Fe 4 S 4 ] clusters. b Representative Coomassie staining of purified nsp14 and nsp10, individually or co-expressed and purified from Expi293F cells ( n = 4 independent experiments). c Representative Coomassie staining of purified nsp14 and nsp10, individually or co-expressed in E. coli ( n = 3 independent experiments). d Images of columns during the purification of nsp14 from Expi293F and E. coli cells are shown on the left. Eluates of purified nsp14 proteins, exhibiting the typical brown color characteristic of Fe-S enzymes, are presented on the right. e Continuous-wave EPR (CW-EPR) spectrum recorded at 10 K for nsp14 and nsp10 upon reduction with sodium dithionite (DT). The parameters obtained by simulating the spectrum of the reduced clusters in nsp14 are ( g ) = (2.05, 1.92, 1.86) and for nsp10 are ( g ) = (2.05, 1.90, 1.86). f Inductively coupled plasma mass spectrometry (ICP-MS) performed on nsp14 and nsp10 wild-type (WT) and variants, as indicated ( n = 3 independent experiments). Nsp14 Aero* represents nsp14 purified under standard oxygen tension with (+Zn) or without zinc reconstitution. Nsp10 Aero* represents nsp10 purified under standard oxygen tension (on the bench) without zinc reconstitution. Nsp14/nsp10 Aero represents the heterodimeric complex of the two proteins co-expressed and purified from mammalian cells without zinc reconstitution. Data are presented as mean values ± SD. g Table summarizing the iron and zinc content of nsp14 and nsp10 WT and variant proteins, as determined by ICP-MS. The standard deviation (SD) was calculated from three independent experiments. h Metal centers coordinating zinc in the available structure of nsp14 (PDB ID: 7N0C). Two sites identified as Fe-S cluster ligating centers (Cys 3 His) are highlighted, with a cubane cluster depicted beneath the box for illustrative purposes. i Metal centers coordinating zinc in the available structure of nsp10 (PDB ID: 7N0C). Both sites were identified as Fe-S centers in the present study. Source data are provided as a Source Data file and in the Supplementary Information.
Article Snippet: Continuous-wave EPR (CW-EPR) spectra were acquired on a Bruker ELEXSYS-II E580 X-band spectrometer.
Techniques: Purification, Staining, Clinical Proteomics, Mass Spectrometry, Variant Assay, Standard Deviation
![a UV-visible absorption spectra of nsp10 and nsp14, individually or co-expressed and purified from Expi293F cells, showing peaks at ~420 nm characteristic of [Fe 4 S 4 ] clusters. b Representative Coomassie staining of purified nsp14 and nsp10, individually or co-expressed and purified from Expi293F cells ( n = 4 independent experiments). c Representative Coomassie staining of purified nsp14 and nsp10, individually or co-expressed in E. coli ( n = 3 independent experiments). d Images of columns during the purification of nsp14 from Expi293F and E. coli cells are shown on the left. Eluates of purified nsp14 proteins, exhibiting the typical brown color characteristic of Fe-S enzymes, are presented on the right. <t>e</t> <t>Continuous-wave</t> <t>EPR</t> <t>(CW-EPR)</t> spectrum recorded at 10 K for nsp14 and nsp10 upon reduction with sodium dithionite (DT). The parameters obtained by simulating the spectrum of the reduced clusters in nsp14 are ( g ) = (2.05, 1.92, 1.86) and for nsp10 are ( g ) = (2.05, 1.90, 1.86). f Inductively coupled plasma mass spectrometry (ICP-MS) performed on nsp14 and nsp10 wild-type (WT) and variants, as indicated ( n = 3 independent experiments). Nsp14 Aero* represents nsp14 purified under standard oxygen tension with (+Zn) or without zinc reconstitution. Nsp10 Aero* represents nsp10 purified under standard oxygen tension (on the bench) without zinc reconstitution. Nsp14/nsp10 Aero represents the heterodimeric complex of the two proteins co-expressed and purified from mammalian cells without zinc reconstitution. Data are presented as mean values ± SD. g Table summarizing the iron and zinc content of nsp14 and nsp10 WT and variant proteins, as determined by ICP-MS. The standard deviation (SD) was calculated from three independent experiments. h Metal centers coordinating zinc in the available structure of nsp14 (PDB ID: 7N0C). Two sites identified as Fe-S cluster ligating centers (Cys 3 His) are highlighted, with a cubane cluster depicted beneath the box for illustrative purposes. i Metal centers coordinating zinc in the available structure of nsp10 (PDB ID: 7N0C). Both sites were identified as Fe-S centers in the present study. Source data are provided as a Source Data file and in the Supplementary Information.](https://pub-med-central-images-cdn.bioz.com/pub_med_central_ids_ending_with_6973/pmc12356973/pmc12356973__41467_2025_62832_Fig2_HTML.jpg)