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- EMDB-44585: Cryo-EM structure of NINJ1 K45Q bound to Nb538 -

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Basic information

Entry
Database: EMDB / ID: EMD-44585
TitleCryo-EM structure of NINJ1 K45Q bound to Nb538
Map data
Sample
  • Complex: Complex of NINJ1 with Nb538
    • Protein or peptide: Ninjurin-1
    • Protein or peptide: Nb538
KeywordsInactive-state / Membrane rupture / Complex / MEMBRANE PROTEIN
Function / homology
Function and homology information


pericyte cell migration / ferroptosis / pyroptotic cell death / positive regulation of osteoclast development / cell adhesion mediator activity / membrane destabilizing activity / leukocyte chemotaxis involved in inflammatory response / cytolysis / hyaloid vascular plexus regression / positive regulation of toll-like receptor 4 signaling pathway ...pericyte cell migration / ferroptosis / pyroptotic cell death / positive regulation of osteoclast development / cell adhesion mediator activity / membrane destabilizing activity / leukocyte chemotaxis involved in inflammatory response / cytolysis / hyaloid vascular plexus regression / positive regulation of toll-like receptor 4 signaling pathway / cell-cell adhesion mediator activity / tissue regeneration / programmed necrotic cell death / muscle cell differentiation / cellular hyperosmotic response / filopodium membrane / heterotypic cell-cell adhesion / positive regulation of cell-matrix adhesion / macrophage chemotaxis / regulation of angiogenesis / lipopolysaccharide binding / synaptic membrane / protein homooligomerization / positive regulation of inflammatory response / angiogenesis / killing of cells of another organism / cell adhesion / inflammatory response / extracellular region / plasma membrane
Similarity search - Function
Biological speciesMus musculus (house mouse) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsPourmal S / Johnson MC / Deshpande I
Funding support1 items
OrganizationGrant numberCountry
Other private
CitationJournal: Nature / Year: 2025
Title: Autoinhibition of dimeric NINJ1 prevents plasma membrane rupture.
Authors: Sergei Pourmal / Melissa E Truong / Matthew C Johnson / Ying Yang / Lijuan Zhou / Kamela Alegre / Irma B Stowe / Shalini Gupta / Phoebe A Chen / Yingnan Zhang / Alexis Rohou / Kim Newton / ...Authors: Sergei Pourmal / Melissa E Truong / Matthew C Johnson / Ying Yang / Lijuan Zhou / Kamela Alegre / Irma B Stowe / Shalini Gupta / Phoebe A Chen / Yingnan Zhang / Alexis Rohou / Kim Newton / Nobuhiko Kayagaki / Vishva M Dixit / Ishan Deshpande /
Abstract: Lytic cell death culminates in plasma membrane rupture, which releases large intracellular molecules to augment the inflammatory response. Plasma membrane rupture is mediated by the effector membrane ...Lytic cell death culminates in plasma membrane rupture, which releases large intracellular molecules to augment the inflammatory response. Plasma membrane rupture is mediated by the effector membrane protein ninjurin-1 (NINJ1), which polymerizes and ruptures the membrane via its hydrophilic face. How NINJ1 is restrained under steady-state conditions to ensure cell survival remains unknown. Here we describe the molecular underpinnings of NINJ1 inhibition. Using cryogenic electron microscopy, we determined the structure of inactive-state mouse NINJ1 bound to the newly developed nanobody Nb538. Inactive NINJ1 forms a face-to-face homodimer by adopting a three-helix conformation with unkinked transmembrane helix 1 (TM1), in contrast to the four-helix TM1-kinked active conformation. Accordingly, endogenous NINJ1 from primary macrophages is a dimer under steady-state conditions. Inactive dimers sequester the membrane rupture-inducing hydrophilic face of NINJ1 and occlude the binding site for kinked TM1 from neighbouring activated NINJ1 molecules. Mutagenesis studies in cells show that destabilization of inactive face-to-face dimers leads to NINJ1-mediated cell death, whereas stabilization of face-to-face dimers inhibits NINJ1 activity. Moreover, destabilizing mutations prompt spontaneous TM1 kink formation, a hallmark of NINJ1 activation. Collectively, our data demonstrate that dimeric NINJ1 is autoinhibited in trans to prevent unprovoked plasma membrane rupture and cell death.
History
DepositionApr 23, 2024-
Header (metadata) releaseOct 16, 2024-
Map releaseOct 16, 2024-
UpdateApr 16, 2025-
Current statusApr 16, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44585.png

Downloads & links

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Map

FileDownload / File: emd_44585.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 288 pix.
= 210.528 Å		0.73 Å/pix.
x 288 pix.
= 210.528 Å		0.73 Å/pix.
x 288 pix.
= 210.528 Å

Surface

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.731 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.056
Minimum - Maximum-0.3001126 - 0.49631074
Average (Standard dev.)0.0001890534 (±0.011506472)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 210.528 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_44585_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_44585_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of NINJ1 with Nb538

EntireName: Complex of NINJ1 with Nb538
Components
  • Complex: Complex of NINJ1 with Nb538
    • Protein or peptide: Ninjurin-1
    • Protein or peptide: Nb538

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Supramolecule #1: Complex of NINJ1 with Nb538

SupramoleculeName: Complex of NINJ1 with Nb538 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Ninjurin-1

MacromoleculeName: Ninjurin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 18.648311 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MESGTEEYEL NGDLRPGSPG SPDALPPRWG LRNRPINVNH YANKQSAAES MLDIALLMAN ASQLKAVVEQ GNDFAFFVPL VVLISISLV LQIGVGVLLI FLVKYDLNNP AKHAKLDFLN NLATGLVFII VVVNIFITAF GVQKPVMDVA PRQGSENLYF Q GSDYKDDD DK

UniProtKB: Ninjurin-1

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Macromolecule #2: Nb538

MacromoleculeName: Nb538 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 15.126499 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
EVQLVESGGG LVQPGGSLRL SCAASGFNVY SSSYYYVGWV RRAPGKGEEL VARISPSYGY TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC EVYIFGQYFE SGQGTLVTVS SDKTHTGGSS GGSHHHHHHG S

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 102877
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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