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- EMDB-15087: murine perforin-2 pore on liposome by subtomogram averaging -

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Basic information

Entry
Database: EMDB / ID: EMD-15087
Titlemurine perforin-2 pore on liposome by subtomogram averaging
Map datamain map, low pass filtered to 18 Angstrom
Sample
  • Complex: murine perforin-2 (Mpeg1) ectodomain pore
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 18.0 Å
AuthorsYu X / Ni T / Zhang P / Gilbert R
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust United Kingdom
CitationJournal: EMBO J / Year: 2022
Title: Cryo-EM structures of perforin-2 in isolation and assembled on a membrane suggest a mechanism for pore formation.
Authors: Xiulian Yu / Tao Ni / George Munson / Peijun Zhang / Robert J C Gilbert /
Abstract: Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a ...Perforin-2 (PFN2, MPEG1) is a key pore-forming protein in mammalian innate immunity restricting intracellular bacteria proliferation. It forms a membrane-bound pre-pore complex that converts to a pore-forming structure upon acidification; but its mechanism of conformational transition has been debated. Here we used cryo-electron microscopy, tomography and subtomogram averaging to determine structures of PFN2 in pre-pore and pore conformations in isolation and bound to liposomes. In isolation and upon acidification, the pre-assembled complete pre-pore rings convert to pores in both flat ring and twisted conformations. On membranes, in situ assembled PFN2 pre-pores display various degrees of completeness; whereas PFN2 pores are mainly incomplete arc structures that follow the same subunit packing arrangements as found in isolation. Both assemblies on membranes use their P2 β-hairpin for binding to the lipid membrane surface. Overall, these structural snapshots suggest a molecular mechanism for PFN2 pre-pore to pore transition on a targeted membrane, potentially using the twisted pore as an intermediate or alternative state to the flat conformation, with the capacity to cause bilayer distortion during membrane insertion.
History
DepositionJun 2, 2022-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateDec 14, 2022-
Current statusDec 14, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15087.png

Downloads & links

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Map

FileDownload / File: emd_15087.map.gz / Format: CCP4 / Size: 95 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmain map, low pass filtered to 18 Angstrom
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.63 Å/pix.
x 292 pix.
= 475.96 Å		1.63 Å/pix.
x 292 pix.
= 475.96 Å		1.63 Å/pix.
x 292 pix.
= 475.96 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.63 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.332
Minimum - Maximum-0.70894617 - 1.1522648
Average (Standard dev.)0.009449336 (±0.090463)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions292292292
Spacing292292292
CellA=B=C: 475.96 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15087_msk_1.map
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Half map: #1

Fileemd_15087_half_map_1.map
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Half map: #2

Fileemd_15087_half_map_2.map
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Sample components

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Entire : murine perforin-2 (Mpeg1) ectodomain pore

EntireName: murine perforin-2 (Mpeg1) ectodomain pore
Components
  • Complex: murine perforin-2 (Mpeg1) ectodomain pore

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Supramolecule #1: murine perforin-2 (Mpeg1) ectodomain pore

SupramoleculeName: murine perforin-2 (Mpeg1) ectodomain pore / type: complex / Chimera: Yes / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 3.8
VitrificationCryogen name: ETHANE
Detailspurified perforin-2 ecto domain protein was added to liposomes at pH3.8

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 123.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 6.0 µm / Nominal defocus min: 3.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: emClarity, RELION) / Number subtomograms used: 992
ExtractionNumber tomograms: 141 / Number images used: 1369
Final angle assignmentType: OTHER / Details: cross correlation

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