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- EMDB-15076: murine perforin-2 (Mpeg1) prepore on membrane by cryoET subtomogr... -

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Basic information

Entry
Database: EMDB / ID: EMD-15076
Titlemurine perforin-2 (Mpeg1) prepore on membrane by cryoET subtomogram averaging
Map datafinal combined map
Sample
  • Complex: murine perforin-2 ectodomain prepore assembled on liposome
Biological speciesMus musculus (house mouse)
Methodsubtomogram averaging / cryo EM / Resolution: 6.0 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 1, 2022-
Header (metadata) releaseAug 17, 2022-
Map releaseAug 17, 2022-
UpdateDec 14, 2022-
Current statusDec 14, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15076.png

Downloads & links

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Map

FileDownload / File: emd_15076.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfinal combined map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.88 Å/pix.
x 240 pix.
= 450. Å		1.88 Å/pix.
x 240 pix.
= 450. Å		1.88 Å/pix.
x 240 pix.
= 450. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.875 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 56.1
Minimum - Maximum-104.1329 - 230.56639
Average (Standard dev.)3.9707082e-10 (±16.045284)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 450.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15076_msk_1.map
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Half map: #1

Fileemd_15076_half_map_1.map
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Half map: #2

Fileemd_15076_half_map_2.map
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Sample components

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Entire : murine perforin-2 ectodomain prepore assembled on liposome

EntireName: murine perforin-2 ectodomain prepore assembled on liposome
Components
  • Complex: murine perforin-2 ectodomain prepore assembled on liposome

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Supramolecule #1: murine perforin-2 ectodomain prepore assembled on liposome

SupramoleculeName: murine perforin-2 ectodomain prepore assembled on liposome
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
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: 7.5
VitrificationCryogen name: ETHANE
Detailspurified murine perforin-2 ectodomain were incubated with liposomes at pH7.5

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / 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: 5.0 µm / Nominal defocus min: 2.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C16 (16 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 3695
ExtractionNumber tomograms: 30 / Number images used: 25530
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER

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