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- EMDB-20620: EM structure of MPEG-1 (L425K, alpha conformation) soluble pre-po... -

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Basic information

Entry
Database: EMDB / ID: EMD-20620
TitleEM structure of MPEG-1 (L425K, alpha conformation) soluble pre-pore complex
Map dataMPEG-1 (L425K, alpha conformation) soluble pre-pore complex
Sample
  • Complex: MPEG-1 (L425K, alpha conformation) soluble pre-pore
    • Protein or peptide: Macrophage-expressed gene 1 protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Function / homology
Function and homology information


dendritic cell antigen processing and presentation / antigen processing and presentation of exogenous peptide antigen / phagolysosome membrane / antibacterial innate immune response / wide pore channel activity / antigen processing and presentation of exogenous peptide antigen via MHC class I / phagocytic vesicle / phagocytic vesicle membrane / cytoplasmic vesicle / defense response to Gram-negative bacterium ...dendritic cell antigen processing and presentation / antigen processing and presentation of exogenous peptide antigen / phagolysosome membrane / antibacterial innate immune response / wide pore channel activity / antigen processing and presentation of exogenous peptide antigen via MHC class I / phagocytic vesicle / phagocytic vesicle membrane / cytoplasmic vesicle / defense response to Gram-negative bacterium / adaptive immune response / defense response to Gram-positive bacterium / defense response to bacterium / extracellular region
Similarity search - Function
Macrophage-expressed gene 1 protein / membrane-attack complex / perforin / Membrane attack complex/perforin (MACPF) domain profile. / MAC/Perforin domain / Membrane attack complex component/perforin (MACPF) domain
Similarity search - Domain/homology
Macrophage-expressed gene 1 protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.37 Å
AuthorsPang SS / Bayly-Jones C
Funding support Australia, 1 items
OrganizationGrant numberCountry
Australian Research Council (ARC) Australia
CitationJournal: Nat Commun / Year: 2019
Title: The cryo-EM structure of the acid activatable pore-forming immune effector Macrophage-expressed gene 1.
Authors: Siew Siew Pang / Charles Bayly-Jones / Mazdak Radjainia / Bradley A Spicer / Ruby H P Law / Adrian W Hodel / Edward S Parsons / Susan M Ekkel / Paul J Conroy / Georg Ramm / Hariprasad ...Authors: Siew Siew Pang / Charles Bayly-Jones / Mazdak Radjainia / Bradley A Spicer / Ruby H P Law / Adrian W Hodel / Edward S Parsons / Susan M Ekkel / Paul J Conroy / Georg Ramm / Hariprasad Venugopal / Phillip I Bird / Bart W Hoogenboom / Ilia Voskoboinik / Yann Gambin / Emma Sierecki / Michelle A Dunstone / James C Whisstock /
Abstract: Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, ...Macrophage-expressed gene 1 (MPEG1/Perforin-2) is a perforin-like protein that functions within the phagolysosome to damage engulfed microbes. MPEG1 is thought to form pores in target membranes, however, its mode of action remains unknown. We use cryo-Electron Microscopy (cryo-EM) to determine the 2.4 Å structure of a hexadecameric assembly of MPEG1 that displays the expected features of a soluble prepore complex. We further discover that MPEG1 prepore-like assemblies can be induced to perforate membranes through acidification, such as would occur within maturing phagolysosomes. We next solve the 3.6 Å cryo-EM structure of MPEG1 in complex with liposomes. These data reveal that a multi-vesicular body of 12 kDa (MVB12)-associated β-prism (MABP) domain binds membranes such that the pore-forming machinery of MPEG1 is oriented away from the bound membrane. This unexpected mechanism of membrane interaction suggests that MPEG1 remains bound to the phagolysosome membrane while simultaneously forming pores in engulfed bacterial targets.
History
DepositionAug 20, 2019-
Header (metadata) releaseSep 25, 2019-
Map releaseSep 25, 2019-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0251
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0251
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6u2j
  • Surface level: 0.0251
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20620.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMPEG-1 (L425K, alpha conformation) soluble pre-pore complex
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.0251 / Movie #1: 0.0251
Minimum - Maximum-0.17859133 - 0.40128154
Average (Standard dev.)0.00013969785 (±0.007847384)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 423.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z424.000424.000424.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.1790.4010.000

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Supplemental data

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Mask #1

Fileemd_20620_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: MPEG-1 (L425K, alpha conformation) soluble pre-pore complex unsharpened...

Fileemd_20620_additional.map
AnnotationMPEG-1 (L425K, alpha conformation) soluble pre-pore complex unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: MPEG-1 (L425K, alpha conformation) soluble pre-pore complex, half...

Fileemd_20620_half_map_1.map
AnnotationMPEG-1 (L425K, alpha conformation) soluble pre-pore complex, half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: MPEG-1 (L425K, alpha conformation) soluble pre-pore complex, half...

Fileemd_20620_half_map_2.map
AnnotationMPEG-1 (L425K, alpha conformation) soluble pre-pore complex, half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : MPEG-1 (L425K, alpha conformation) soluble pre-pore

EntireName: MPEG-1 (L425K, alpha conformation) soluble pre-pore
Components
  • Complex: MPEG-1 (L425K, alpha conformation) soluble pre-pore
    • Protein or peptide: Macrophage-expressed gene 1 protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: MPEG-1 (L425K, alpha conformation) soluble pre-pore

SupramoleculeName: MPEG-1 (L425K, alpha conformation) soluble pre-pore / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
Molecular weightTheoretical: 2.3 MDa

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Macromolecule #1: Macrophage-expressed gene 1 protein

MacromoleculeName: Macrophage-expressed gene 1 protein / type: protein_or_peptide / ID: 1 / Number of copies: 16 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 71.126148 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: KSGKPSGEMD EVGVQKCKNA LKLPVLEVLP GGGWDNLRNV DMGRVMELTY SNCRTTEDGQ YIIPDEIFTI PQKQSNLEMN SEILESWAN YQSSTSYSIN TELSLFSKVN GKFSTEFQRM KTLQVKDQAI TTRVQVRNLV YTVKINPTLE LSSGFRKELL D ISDRLENN ...String:
KSGKPSGEMD EVGVQKCKNA LKLPVLEVLP GGGWDNLRNV DMGRVMELTY SNCRTTEDGQ YIIPDEIFTI PQKQSNLEMN SEILESWAN YQSSTSYSIN TELSLFSKVN GKFSTEFQRM KTLQVKDQAI TTRVQVRNLV YTVKINPTLE LSSGFRKELL D ISDRLENN QTRMATYLAE LLVLNYGTHV TTSVDAGAAL IQEDHLRASF LQDSQSSRSA VTASAGLAFQ NTVNFKFEEN YT SQNVLTK SYLSNRTNSR VQSIGGVPFY PGITLQAWQQ GITNHLVAID RSGLPLHFFI NPNMLPDLPG PLVKKVSKTV ETA VKRYYT FNTYPGCTDL NSPNFNFQAN TDDGSCEGKM TNFSFGGVYQ ECTQLSGNRD VLLCQKLEQK NPLTGDFSCP SGYS PVHLL SQIHEEGYNH LECHRKCTLK VFCKTVCEDV FQVAKAEFRA FWCVASSQVP ENSGLLFGGL FSSKSINPMT NAQSC PAGY FPLRLFENLK VCVSQDYELG SRFAVPFGGF FSCTVGNPLV DPAISRDLGA PSLKKCPGGF SQHPALISDG CQVSYC VKS GLFTGGSLPP ARLPPFTRPP LMSQAATNTV IVTNSENARS WIKDSQTHQW RLGEPIELRR AMNVIHGDGG GLSHHHH HH

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 16 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration2 mg/mL
BufferpH: 7.2
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 44.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C16 (16 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.37 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 138422
FSC plot (resolution estimation)

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