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. 2022 Feb 19;121(7):1289–1298. doi: 10.1016/j.bpj.2022.02.026

Figure 2.

Figure 2

Deciphering the molecular organization of Get4-Get5 complex. (A) Mass spectrum of the Get4-Get5 heterotetramer: (Get4)2(Get5)2. Scheme I and Scheme II show the possible organization of the complex. Scheme I: the complex is formed only via the Get5/5 interface, Scheme II: complex is formed via Get5/5 and Get4/4 interface. The shorter peaks visible in the mass spectrum were formed by the complex with one truncated form of the Get4 lacking the first two residues (Met-Val), and the higher peaks were formed by complex with two full-length Get4. Inset shows the two peaks for the representative 23+ charge state. (B) MS/MS spectra of (Get4)2(Get5)2. CID of the (Get4)2(Get5)2 produced two sets of subcomplexes. Peaks in purple highlight the subcomplexes generated by the loss of a monomeric Get4 unit from the overall complex. The remainder of the trimer Get4-(Get5)2 species was observed at the higher m/z range (purple peaks in the right panel). The lost, monomeric Get4 molecule was detected in the lower m/z region (purple peaks in the left panel). Peaks in blue highlight the subcomplexes generated by the loss of a monomeric Get5 unit from the overall complex. The remainder of the trimer (Get4)2-Get5 species was observed at the higher m/z range (purple peaks in the right panel). The lost, monomeric Get5 molecule was detected in the lower m/z region (blue peaks in the left panel). Similar to panel A, subcomplexes containing Get4 showed a small amount of truncated Get4 containing population. (C) Deciphering the molecular organization of (Get4)2(Get5)2. Ions owing to the subcomplex (Get4) (Get5)2 and Get4 can be produced by CID of parent ions with molecular organization presented in both Schemes I and II. In contrast, ions owing to dissociated subcomplex (Get4)2(Get5) and Get5 can be produced by CID of parent ions with molecular organization presented only in Scheme II. Dotted lines on the structure indicate interaction interfaces dissociated by CID during the generation of daughter ions. Similar spectra were obtained from three independent protein preparations.