Figure 3.
MS Analysis of the CSM Complex Establishing Its Composition, Subunit Connectivity, and crRNA Binding
(A) MS spectrum of the intact CSM reveals a well-resolved charge-state series at 8,500 m/z with a molecular mass of 427,789 Da, 122 kDa higher than the expected mass for a stoichiometric complex comprising eight subunits and one crRNA.
(B) The 49+ charge state of the complex was selected and subjected to acceleration, and dissociation of subunits Sso1424, Sso1428, and Sso1426/7 was observed by tandem MS.
(C) The molar ratio of Sso1426:Sso1428 was determined as 4:1 by relative quantification of tryptic peptides of Sso1426 and Sso1428 (GSVDLNYLR and FLDSLPISYSLNTR, respectively; see Table 1 and Table S3). Labeled peptides of the same sequences were synthesized and used as reference. (15N,13C)-labeled residues are colored red.
(D–F) Disassembly of the CSM complex resulted in a series of subcomplexes (i–v) in solutions of decreasing pH: 3.9 (D), 3.5 (E), and 3.2 (F).
(G) A complete CSM subunit interaction map was derived from MS data, including intact subcomplexes, crosslinking, and quantitative analysis (see also Figures S1–S3 and Tables S2–S6). The crRNA binds to subunits making up the major backbone and dissociates together with three copies of Sso1426 and Sso1432.