The cryo-EM structure of deactive Y. lipolytica complex I [Protein Data Bank (PDB) ID 6GCS] was used as template for building the wild-type complex I in COOT (53). Full-atom models of all accessory subunits were built. A previously unidentified density with one TM helix fitted the sequence of NUUM, a subunit without homolog in mammalian complex I. The density assigned as NUUM in the previous map (8) occupies the position of the mammalian subunit NDUFB2. A BLAST search in fungi yielded several homologs, including one in the yeast Pichia pastoris (Komatagaella phaffii). A second BLAST search with this yeast homolog against the Y. lipolytica proteome ( yielded the sequence of YALI1_D3003g, which fitted the density. We refer to this previously unidentified Y. lipolytica complex I subunit as NIGM. Lipids were built wherever possible, and the fatty acid tails truncated to the visible density. Two LMNG molecules were identified and built. The structure was refined using phenix.real_space_refine (54), followed by several rounds of rebuilding in COOT. The ΔNDUFS4 and ΔNDUFS6 structures were built with the wild-type model as template. The model was cross-validated against overfitting by the method as described earlier (55): The FSC between the refined model and the final map was determined (FSCSUM; fig. S2B). Random shifts (up to 0.3 Å) were introduced into the coordinates of the final model, followed by refinement in PHENIX against the first unfiltered half-map. Then, the FSC was determined between this refined model and the half-map used for refinement (FSCwork), and the FSC against the second half-map, which was not used at any point during refinement (FSCfree). The marginal gap between the curves describing FSCwork and FSCfree indicates that the model was not overfitted (see fig. S2B). A quality check indicated excellent stereochemistry with 91.51 to 93.89% of the nonglycine and nonproline residues found in the most-favored regions and with 0.15 to 0.17% outliers (all-atom clashscore, 8.36 to 9.29). Refinement and validation statistics were summarized in Table 1. Figures were drawn with Chimera (56) and PyMOL (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger, LLC).

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