Human Mitochondrial Barrel Factory Caught in Two States by Cryo-EM

Deep inside every human cell, mitochondria are constantly importing the protein building blocks they need to function, and a molecular machine called the sorting and assembly machinery, or SAM, is responsible for folding and inserting one of the most difficult classes of proteins in biology: beta-barrel proteins. These proteins, which include TOM40, the voltage-dependent anion channels known as VDACs, and the SAM core component SAM50 itself, must be threaded across the outer mitochondrial membrane and then folded into precise barrel shapes within the greasy lipid environment of that membrane. Now, a team of researchers led by Ping Yin at Huazhong Agricultural University in Wuhan, China, has captured the human SAM complex in unprecedented detail, revealing both a monomeric and a dimeric architecture that together offer the clearest picture yet of how human mitochondria build their own barrel-shaped gatekeepers. The structures, determined by cryo-electron microscopy and published in Cell Research, provide the first direct structural evidence that the barrel-switching mechanism long observed in fungal systems also operates in human cells, but with distinctly mammalian twists.

The importance of beta-barrel proteins to mitochondrial biology is difficult to overstate. TOM40 forms the primary channel through which nearly all mitochondrial proteins enter the organelle, while the VDACs regulate the flow of metabolites such as ATP and ions across the outer membrane. SAM50, the central pore-forming subunit of the SAM complex, belongs to the Omp85 family of bacterial and organellar proteins, and it carries a distinctive structural feature called a lateral gate. This gate is a region of the barrel that can open sideways, allowing a nascent beta-barrel substrate to partially release from the SAM50 lumen and begin folding into its own barrel within the membrane. Decades of work on bacterial and mitochondrial assembly machineries have established that barrel folding proceeds hand in hand with conformational changes at these lateral gates, making the gate a dynamic control point rather than a static seam.

In fungi, where the SAM complex consists of Sam50 plus the peripheral subunits Sam35 and Sam37, high-resolution structural studies have established what researchers call a beta-barrel-switching model. In this model, a second beta-barrel protein binds dynamically at a site immediately adjacent to the lateral gate of Sam50, effectively handing off the growing substrate from one barrel to another as folding proceeds. The fungal structures showed this switching site occupied by barrel proteins caught in the act of assembly, providing a snapshot of the folding pathway in progress. But a critical question has lingered: do mammalian SAM complexes, which use entirely different peripheral subunits, work the same way? In humans, the proteins METAXIN1 and METAXIN2 replace Sam37 and Sam35 respectively, and whether the human machine employs an equivalent switching mechanism had remained unresolved.

The new study answers that question with striking structural detail. The research team, including co-first authors Ling Yan, Zeyuan Guan and Qiang Wang, determined cryo-EM structures of two distinct forms of the human SAM complex. The first is a monomeric complex containing a single SAM50 molecule paired with one METAXIN2, forming a SAM50-METAXIN2 core. The second is a dimeric complex containing two SAM50 molecules, again with one METAXIN2 associated. The atomic coordinates and cryo-EM maps for both structures have been deposited in public databases, with the monomeric complex archived under PDB code 25WG and EMDB entry EMD-80424, and the dimeric complex under PDB 25WF and EMD-80423, allowing researchers worldwide to examine the molecular details directly.

The dimeric structure is the showstopper. Rather than simply sitting side by side, the two SAM50 barrels adopt what the authors describe as an interlocked conformation: each SAM50 molecule extends beta-strands into the barrel lumen of its partner. This molecular intertwining means that one SAM50 barrel physically occupies the beta-barrel-switching site adjacent to the lateral gate of the other SAM50. In other words, the dimer captures the human SAM complex in a configuration that directly mirrors the fungal beta-barrel-switching model, but with the remarkable twist that the second barrel at the switching site is another copy of SAM50 itself. This suggests that in human mitochondria, a second SAM50 molecule may serve as a placeholder or scaffold at the switching site, potentially priming the machine for substrate engagement or reflecting a regulatory state in the assembly cycle.

To test whether this dimeric arrangement is functionally meaningful rather than a crystallographic artifact of sample preparation, the team turned to biochemical and cell-based approaches. Crosslinking experiments, which chemically freeze transient protein-protein contacts so they can be detected, provided independent evidence for the close apposition of the two SAM50 molecules in living systems. More tellingly, the researchers employed fluorescence lifetime imaging microscopy combined with Foerster resonance energy transfer, a technique known as FLIM-FRET that reports on nanometer-scale distances between fluorescently labeled proteins inside cells. These analyses revealed that METAXIN1, the mammalian counterpart of fungal Sam37, alters the conformation of SAM50 and changes the relative arrangement of the two SAM50 molecules in the dimeric complex.

This METAXIN1-dependent remodeling is a key finding because it demonstrates that the dimeric SAM assembly is not a rigid, static structure but a dynamic platform whose geometry is actively tuned by peripheral subunits. In the fungal system, Sam37 plays roles in substrate release from the SAM complex, and the implication is that METAXIN1 may perform an analogous regulatory function in human cells, repositioning the SAM50 barrels to facilitate the progression of substrate folding and release. The structural and functional data together suggest a working model in which the human SAM complex cycles between monomeric and dimeric states, with METAXIN1 and METAXIN2 orchestrating conformational transitions that drive beta-barrel substrates through the folding pathway.

The evolutionary dimension of this work is equally compelling. The fact that the beta-barrel-switching principle is conserved from fungi to humans, despite the complete replacement of the peripheral subunits Sam35 and Sam37 with METAXIN2 and METAXIN1, indicates that the core mechanism of barrel assembly is ancient and tightly constrained. The Omp85 family spans bacteria, chloroplasts and mitochondria, and lateral-gate dynamics have been documented across all these systems. What the new structures add is the demonstration that the switching site itself, the physical location where a second barrel docks next to the gate, is a conserved feature of the mitochondrial machinery, even as the molecular players that regulate it have been swapped out during evolution. This kind of architectural conservation with component turnover is a recurring theme in mitochondrial biology and underscores the deep common ancestry of the organelle’s protein import systems.

Why should this matter beyond structural biology? Defects in mitochondrial protein import and outer membrane assembly are increasingly linked to human disease, and the VDACs and TOM40 in particular have been implicated in neurodegeneration, cancer metabolism and mitochondrial dysfunction more broadly. By defining the conformational states of the human SAM complex and identifying METAXIN1 as a remodeling factor, the study establishes a framework that other researchers can use to probe how barrel assembly goes wrong in disease contexts, and potentially how it could be modulated. The structures also provide concrete molecular targets for future studies of METAXIN function, which until now have been largely mysterious in mechanistic terms.

The work also showcases the power of modern cryo-electron microscopy to resolve small, dynamic membrane protein assemblies that were once considered beyond reach. Capturing both the monomeric and interlocked dimeric states of the human SAM complex, complete with the beta-strand exchange between barrels, required both technical skill in sample preparation and data collection, acknowledged by the authors to the cryo-EM facilities at the University of Science and Technology of China and Huazhong Agricultural University, and sophisticated computational reconstruction. As cryo-EM continues to push toward smaller and more heterogeneous complexes, studies like this one are filling in the remaining gaps in our understanding of how cells build their most essential molecular machines. For the mitochondrial outer membrane, the picture is now coming into focus: a conserved barrel-switching engine, remodeled by mammalian-specific regulators, assembling the channels that keep cellular power plants running.

Subject of Research: Cryo-EM structures of the human mitochondrial SAM complex involved in beta-barrel protein assembly

Article Title: Cryo-EM structures of monomeric and dimeric human SAM complexes

Article References: Yan, L., Guan, Z., Wang, Q., Wu, Y., Qi, L., Huang, C., Zhang, J., Hong, S., Zhuang, J., Huang, R., Zhang, L., Duan, R., Liu, Y., Liu, Z., & Yin, P. (2026). Cryo-EM structures of monomeric and dimeric human SAM complexes. Cell Research. https://doi.org/10.1038/s41422-026-01305-w

Image Credits: AI Generated

DOI: 10.1038/s41422-026-01305-w

Keywords: cryo-EM, mitochondria, SAM complex, SAM50, beta-barrel proteins, METAXIN1, METAXIN2, protein folding, outer mitochondrial membrane, TOM40, VDAC, structural biology