Addendum to: Nature Cell Biology https://doi.org/10.1038/s41556-025-01620-1, published online 21 March 2025.

In our paper, we demonstrated that most prohibitin molecules in human cells localize to mitochondria, where they are markedly enriched in the crista membranes. Using cryo-electron tomography and subtomogram averaging, we showed that the human prohibitin complex adopts a bell-shaped architecture at the mitochondrial inner membrane and proposed an 11-fold arrangement of the complex in situ.

Such an 11-fold arrangement has been confirmed by several recent studies, including high-resolution single-particle cryo-EM analyses of prohibitin complexes, as well as in situ cryo-electron tomography of prohibitin complexes in mitochondria of mouse embryonic fibroblasts. Since we published our original paper, another study using cryo-electron tomography suggested a 12-fold arrangement.

As this report used the same human U2OS cell as our study, we decided to re-evaluate our findings. To this end we revisited our tomography data and manually selected 2,383 particles–substantially more than the 817 used in our original study and comparable to the dataset analyzed by Rose et al. (Extended Data Fig. 1). As described in our original paper and following established standards in the field, these particles were first aligned along the z-axis in Dynamo with subsequent randomization of the rotation angle around the z-axis (function dynamo_table_randomize_azimuth in Dynamo) to avoid preferred orientation of the particles due to missing wedge as the vast majority (>95%) of the prohibitin complexes are visible in the tomograms in side views (Extended Data Fig. 2). Subsequently, their poses were automatically refined in 3D without symmetry imposition using Relion 4.0.2 (Fig. 1A; Extended Data Fig. 3). For the re-evaluation, we then conducted a symmetry analysis by examining the banded intensity profile within the annular region of the map (Fig. 1B–E). After band-pass filtering to isolate features with a periodicity corresponding to between 9 and 18 repetitions over the 360° circumference and inverting the profile so peaks corresponded to higher density (dark pixels in Fig. 1B), we observed 11 distinct peaks. Fourier analysis of the intensity spectrum consistently showed a dominant C11 frequency component, with C10 and C12 components less prominent (Fig. 1E). Thus, in the absence of imposed symmetry, the cryo-EM data strongly support the presence of 11 subunits.