biostudies-literatureReif-Trauttmansdorff TDeutsche ForschungsgemeinschaftDeutsche Forschungsgemeinschaft (German Research Foundation)EC | Horizon 2020 Framework ProgrammeEC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020)133https://www.ebi.ac.uk/biostudies/studies/S-EPMC12774963biostudies-literatureUnknown17(1)Methanogenic archaea emit ~1 Gt of methane annually, impacting global carbon cycling and climate. Central to their energy metabolism is a membrane-bound, sodium-translocating methyltransferase complex: the N⁵-tetrahydromethanopterin:CoM-S-methyltransferase (Mtr). It couples methyl transfer between two methanogen-specific cofactors with sodium ion transport across the membrane, forming the only energy-conserving step in hydrogenotrophic methanogenesis. Here, we present a 2.1 Å single-particle cryo-EM structure of the Mtr complex from Methanosarcina mazei. The structure reveals the organization of all catalytic subunits, embedded archaeal lipids and the sodium-binding site. Most strikingly, we discover MtrI, a previously unannotated small open-reading frame encoded protein ( < 100 aa) found Nature communicationsStructure of the Methanosarcina mazei Mtr complex bound to the oxygen-stress responsive small protein MtrI.PMC12774963RTG 2937SCHU 3364/1-1101075992Kahnt JZimmer EHerdering EPascoa TReif-Trauttmansdorff TBohn SSchmitz RASchuller JMKumar AfalseStructure of the Methanosarcina mazei Mtr complex bound to the oxygen-stress responsive small protein MtrI.Methanogenic archaea emit ~1 Gt of methane annually, impacting global carbon cycling and climate. Central to their energy metabolism is a membrane-bound, sodium-translocating methyltransferase complex: the N⁵-tetrahydromethanopterin:CoM-S-methyltransferase (Mtr). It couples methyl transfer between two methanogen-specific cofactors with sodium ion transport across the membrane, forming the only energy-conserving step in hydrogenotrophic methanogenesis. Here, we present a 2.1 Å single-particle cryo-EM structure of the Mtr complex from Methanosarcina mazei. The structure reveals the organization of all catalytic subunits, embedded archaeal lipids and the sodium-binding site. Most strikingly, we discover MtrI, a previously unannotated small open-reading frame encoded protein ( < 100 aa) found 2025-01-01T00:00:00Z2025 Dec2026-07-15T13:28:43.162Z2026-07-05T03:08:35.748ZS-EPMC127749634143673810.1038/s41467-025-67705-5