By structurally characterizing SCs from the S4KO mouse model of Leigh syndrome we show: first, that CI is co-operatively assembled by CIII2; second, that in the absence of NDUFS4 aberrant assembly intermediates accumulate; and third, that it is these intermediates, rather than merely a lack of fully assembled CI, that are pathophysiological. This evidence concerns the gene NDUFS4 and Leigh syndrome.