cryo em density map  (Databank Inc)

Journal: Cell reports

Article Title: Distinct immunity protein families mediate compartment-specific neutralization of a bacterial toxin

doi: 10.1016/j.celrep.2025.116459

Figure Lengend Snippet: (A) Schematic diagram of the S. aureus ess locus from strains ST398 (top) and NCTC8325 (bottom). Genes are color coded by protein family. (B) AlphaFold model of LapX3 (SAPIG0303), LapX4 (SAPIG0304), and EsxX 1-220 . The EsxX “FxG” and the LapX3 “FxxxD” targeting motifs are highlighted in orange. Model confidence metrics are shown in . (C) Top: schematic diagram of the EsxX protein; the glycine zipper motif is shaded light gray. Bottom: amino acid sequence of the EsxX glycine zipper with motif-defining amino acids in red. (D) Left: overnight cultures of S. aureus strain 10.1252.XΔess carrying empty pRab11 (VC) or pRab11 encoding the indicated proteins were serially diluted and spotted onto TSB plates with or without ATC as indicated. Plates were incubated overnight at 37° C. Right: overnight cultures of E. coli strain MG1655 carrying empty pBAD18-cm (VC) or pBAD18-cm encoding the indicated proteins were serially diluted and spotted onto LB plates containing either 1% D-glucose or 0.02% L-arabinose as indicated. Plates were incubated overnight at 37° C. EsxX CT = residues 321–517. (E) Exponential-phase E. coli MG1655 cultures carrying pBAD18-cm (VC) or pBAD18-SA-PIG0305-CT (EsxX CT ) were collected prior to (unind) and post- (ind) induction with 0.2% L-arabinose for 30 min. Samples were incubated with the membrane-potential-dependent dye DisC 3 (5) and the membrane-impermeant nucleic acid stain Sytox green and imaged by phase contrast and fluorescence microscopy. A control sample carrying pBAD18-cm was treated with polymyxin B for 5 min prior to staining. Data were collected over two independent experiments with n = 120–290 cells per condition, and single-cell fluorescence values are plotted with a line to represent the median. The right-hand image shows representative cell images.

Article Snippet: Cryo-EM density map of EsxX bound to ExiA and ExiB , This paper , Electron Microscopy DataBank accession code EMD-72654; https://www.ebi.ac.uk/emdb/EMD-72654.

Techniques: Sequencing, Incubation, Membrane, Staining, Fluorescence, Microscopy, Control

Journal: Cell reports

Article Title: Distinct immunity protein families mediate compartment-specific neutralization of a bacterial toxin

doi: 10.1016/j.celrep.2025.116459

Figure Lengend Snippet: (A) Overnight cultures of S. aureus strain 10.1252.XΔess carrying empty pRab11 (VC) or pRab11 encoding the indicated proteins were serially diluted and spotted onto TSB plates with or without ATC as indicated. Plates were incubated overnight at 37° C. (B) Overnight cultures of E. coli strain MG1655 carrying empty pBAD18-cm (VC) or pBAD18-cm encoding the indicated proteins were serially diluted and spotted onto LB plates containing either 1% D-glucose or 0.02% L-arabinose as indicated. Plates were incubated overnight at 37° C. EsxX CT = residues 321–517. (C) ExiAhis and ExiA-ExiBhis were purified from E. coli MG1655 cultures carrying pTrc-307h or pTrc-307-308h by nickel affinity chromatography and then analyzed by SEC using a Superdex 75 column followed by SDS-PAGE with Coomassie staining. On the left is an AlphaFold model of ExiA (SAPIG0307) and ExiB (SAPIG0308). Model confidence metrics are shown in .

Article Snippet: Cryo-EM density map of EsxX bound to ExiA and ExiB , This paper , Electron Microscopy DataBank accession code EMD-72654; https://www.ebi.ac.uk/emdb/EMD-72654.

Techniques: Incubation, Purification, Affinity Chromatography, SDS Page, Staining

Journal: Cell reports

Article Title: Distinct immunity protein families mediate compartment-specific neutralization of a bacterial toxin

doi: 10.1016/j.celrep.2025.116459

Figure Lengend Snippet: (A) A hisEsxX-ExiA-ExiBts trimer and a LapX3-LapX4-hisEsxX-ExiA-ExiBts pentamer were purified by sequential nickel and Strep-Tactin affinity chromatography and then analyzed by SEC using a Superdex 200 increase column and by SDS-PAGE with Coomassie staining. (B) Representative cryo-EM 2D class averages of EsxX-ExiA-ExiB. Yellow scale bar, 100 Å. (C) Cryo-EM reconstruction of EsxX-ExiA-ExiB, contoured to a threshold of 0.54. Black scale bar, 25 Å. (D) AlphaFold model of EsxX 448-479 -ExiA-ExiB fit to cryo-EM density (cross-correlation of 0.6) of the full map (left) or partial map covering EsxX 448-479 -ExiA-ExiB (right). (E) Close-up view of modeled EsxX 448-479 -ExiB fit into cryo-EM density, colored by subunit (left) or by predicted local distance difference test (pLDDT) score (right). EsxX 448-479 -ExiB contacts within 4 Å (Cα-Cα) are displayed as pseudobonds and colored according to putative alignment error (PAE) score. (F) Kinetics (left) and steady-state response (right) for ExiAB binding to the EsxX glycine zipper peptide (EsxX 448-476 ), assessed by bilayer interferometry. The calculated binding affinity was 22 μM with 1:1 stoichiometry. (G) Structural alignment of ExiA and ExiB using the matchmaker function of ChimeraX. Overall Cα root-mean-square deviation (RMSD) across all residues is 3.9 Å. (H) Surface lipophilicity of ExiB with bound EsxX 448-479 (left) or ExiA (right) in the same model orientation as (G).

Article Snippet: Cryo-EM density map of EsxX bound to ExiA and ExiB , This paper , Electron Microscopy DataBank accession code EMD-72654; https://www.ebi.ac.uk/emdb/EMD-72654.

Techniques: Purification, Affinity Chromatography, SDS Page, Staining, Cryo-EM Sample Prep, Binding Assay

Journal: Cell reports

Article Title: Distinct immunity protein families mediate compartment-specific neutralization of a bacterial toxin

doi: 10.1016/j.celrep.2025.116459

Figure Lengend Snippet: (A) Overnight cultures of S. aureus strain 10.1252.XΔess carrying empty pRab11 (VC) or pRab11 encoding the indicated proteins were serially diluted and spotted onto TSB plates with or without ATC as indicated. Plates were incubated overnight at 37° C. ssEsxX CT denotes the S. aureus α-hemolysin signal sequence fused to the C-terminal 203 residues of EsxX. (B) Genetic loci of staphylococcal esxX (magenta) and its immunity genes, identified by FlaGs analysis of EsxX, ExiC (SAPIG0306), and ExiB (SAPIG0308) (complete data are shown in – ). Homologous genes are color coded. The strains depicted are as follows, with genome accessions given in parentheses: S. lutrae ATCC700373 ( NZ_CP020773 ), S. agnetis 1379 ( NZ_CP045927 ), S. lugdunensis FDAARGOS_141 ( NZ_CP014022 ), S. schweitzeri NCTC13712 ( NZ_LR134304.1 ), S. edaphicus CCM 8730 ( NZ_MRZN01000025.1 ), S. aureus RN4220 ( NZ_CP101124 ), M. sp. Marseille Q6498 ( NZ_OX267714 ), S. borealis 58-52 (NZ_JABVEF010000003), and M. sciuri SNUC 1345 ( NZ_QYJC01000003 ).

Article Snippet: Cryo-EM density map of EsxX bound to ExiA and ExiB , This paper , Electron Microscopy DataBank accession code EMD-72654; https://www.ebi.ac.uk/emdb/EMD-72654.

Techniques: Incubation, Sequencing

Journal: Cell reports

Article Title: Distinct immunity protein families mediate compartment-specific neutralization of a bacterial toxin

doi: 10.1016/j.celrep.2025.116459

Figure Lengend Snippet: (1) The ExiAB immunity complex binds to the toxin domain of nascent EsxX in the cytoplasm, preventing its membrane insertion. LapX3 and LapX4 assemble with the EsxX LXG domain, forming a rod-shaped transport complex carrying a composite FxG-FxxxD targeting signal. (2) The FxG-FxxxD signal delivers the EsxX pre-secretion complex to the T7SS apparatus at the cytoplasmic membrane. As the rod-shaped complex traverses the secretion channel, ExiAB is stripped from the toxin domain, which is carried through the channel in an unfolded conformation. (3) Following toxin secretion, ExiC and ExiD protect the attacking cell membrane from insertion and/or assembly of the EsxX glycine zipper. (4) The LXG domain and/or Lap proteins might be removed from the toxin, which then targets the competitor cell membrane. (5) The toxin inserts into the target membrane and oligomerizes via its glycine zipper, causing depolarization of the target cell membrane. C, cytoplasm; M, membrane; W, cell wall; +, positively charged ion.

Article Snippet: Cryo-EM density map of EsxX bound to ExiA and ExiB , This paper , Electron Microscopy DataBank accession code EMD-72654; https://www.ebi.ac.uk/emdb/EMD-72654.

Techniques: Membrane