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Response to Comment on “Protein assemblies ejected directly from native membranes yield complexes for mass spectrometry”

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Science  08 Nov 2019:
Vol. 366, Issue 6466, eaax3102
DOI: 10.1126/science.aax3102

Figures

  • Fig. 1 Summary of mitochondrial complexes as derived from masses measured in Chorev et al.

    (A) Proposed structure of complex I missing the N module, based on PDB 5LDW. NDUFS3 is present substoichiometrically, leading to peak splitting and enabling us to assign complex I [inset (i) is the schematic used in our original manuscript]. Subunits that were not present according to mass measurement are shown in gray. (B) Complex III structures as assigned according to mass in our original report (based on PDB 1BGY) shown schematically in (i) and color-coded according to the structure in (ii). The monomer was validated using CID (figure S12 of original manuscript); the additional copy of UQCRB, which was used to identify the complex, is shown in our original schematic (pink). (C) The dimer is assembled from the two monomers (left) together with our original assignments (i), now also color-coded according to subunits in the structure (ii).

  • Fig. 2 Proteomics data for ANT-1 summarized.

    Percent coverage, 78%; predicted mass with methionine removed, 32,836 Da (33,364 including modifications). Only modifications found with high confidence are marked.

  • Fig. 3 ANT-1 is a dimer when ejected from native membranes.

    (A) Structure as derived from molecular dynamics simulations reported previously, and ion mobility collision cross section (CCS) values measured for three different charge states of the dimer, are compared with the calculated value obtained using IMPACT. An average value of 5108 Å2 corresponds closely to the calculated value of 5115 Å2. (B) Tandem MS (m/z = 4151) of the complex (66,391 ± 8 Da) ejected directly from bovine mitochondrial inner membranes yields a subunit mass of 33,195 ± 5 Da. (C and D) Inhibitor binding to the ANT-1 dimer. The upper spectra show the charge states of the ANT-1 dimer prior to addition of inhibitors; the lower spectra show binding of bongkrekic acid and CATr, with measured masses 66,887 ± 15 Da (calculated 66,877) and 67,165 ± 10 Da (calculated 67,161), respectively. Membranes were analyzed under conditions that allow removal of fatty acids, as reported in the original publication.

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