Fig. 3: Identification of ‘Ca. L. ossiferum’ cells in the enrichment culture.

a, Hybridization chain reaction-FISH analysis of the enrichment culture stained with DAPI (cyan) and nucleotide probes targeting the major species of the culture, that is, Lokiarchaea cells (red; the sample on the left was 70× concentrated), bacteria (green) and Methanomicrobiales (purple). The FISH experiments were performed five independent times with similar results. Scale bars, 2 µm. b, Low-magnification 2D cryo-electron micrographs of the three major cell types that were observed after screening of the enrichment culture (n = 2 independent cultures), showing a putative ‘Ca. L. ossiferum’ cell with a round cell body and complex cell protrusions (left), a Gram-negative bacterial cell (middle) and an archaeal cell (right). Scale bars, 1 µm. c, Slices through cryo-tomograms of all three organisms shown in c (slice thickness, 9.02 nm), detailing the characteristic cell envelope architecture of the three species. Putative Lokiarchaea show small and unordered surface densities (sd^a) and complex surface proteins (sd^b) protruding from a single membrane. cm, cytoplasmic membrane; cp, cytoplasm; om, outer membrane; pp, periplasm; sl, surface layer. Scale bars, 100 nm. d, Identification of ‘Ca. L. ossiferum’ by Asgard-specific rRNA structures. Left, a sub-tomogram average (11.7 Å resolution) of ribosomes from cryo-tomograms of putative lokiarchaeal cells (large-subunit proteins (LSU), blue; small-subunit proteins (SSU), orange; rRNA, white). Middle, secondary structure prediction of the ‘Ca. L. ossiferum’ large-subunit rRNA (expansion segments ES9/ES39 are labelled). Right, a superposition of the average with a low-pass filtered (11 Å) map of the T. kodakarensis 70S ribosome (Protein Data Bank (PDB): 6SKF; yellow). The ‘Ca. L. ossiferum’ structure (white) shows prominent additional rRNA features that were identified as the Asgard-specific rRNA expansion segments ES9 and ES39. See also Extended Data Fig. 6.