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- EMDB-20619: EM map of MPEG-1(L425K, alpha conformation) soluble pre-pore complex -

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Basic information

Entry
Database: EMDB / ID: EMD-20619
TitleEM map of MPEG-1(L425K, alpha conformation) soluble pre-pore complex
Map dataMPEG-1(L425K, alpha conformation) soluble pre-pore complex, sharpened map
Sample
  • Complex: Head-to-head assembly of MPEG-1 (L425K, alpha conformation) soluble pre-pore
    • Protein or peptide: Macrophage-Expressed Gene 1 protein
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsPang SS / Bayly-Jones C
Funding support Australia, 1 items
OrganizationGrant numberCountry
Australian Research Council 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 19, 2019-
Header (metadata) releaseSep 25, 2019-
Map releaseSep 25, 2019-
UpdateSep 25, 2019-
Current statusSep 25, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0264
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0264
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20619.png

Downloads & links

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Map

FileDownload / File: emd_20619.map.gz / Format: CCP4 / Size: 149.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMPEG-1(L425K, alpha conformation) soluble pre-pore complex, sharpened map
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.0264 / Movie #1: 0.0264
Minimum - Maximum-0.16101511 - 0.27217007
Average (Standard dev.)0.0009211707 (±0.010345885)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions340340340
Spacing340340340
CellA=B=C: 360.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z340340340
origin x/y/z0.0000.0000.000
length x/y/z360.400360.400360.400
α/β/γ90.00090.00090.000
start NX/NY/NZ1548552
NX/NY/NZ198170217
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS340340340
D min/max/mean-0.1610.2720.001

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Supplemental data

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Mask #1

Fileemd_20619_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_20619_additional.map
AnnotationMPEG-1(L425K, alpha conformation) soluble pre-pore complex, unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_20619_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

Histogram

Histogram (log scale)

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

Fileemd_20619_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

Histogram

Histogram (log scale)

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Sample components

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Entire : Head-to-head assembly of MPEG-1 (L425K, alpha conformation) solub...

EntireName: Head-to-head assembly of MPEG-1 (L425K, alpha conformation) soluble pre-pore
Components
  • Complex: Head-to-head assembly of MPEG-1 (L425K, alpha conformation) soluble pre-pore
    • Protein or peptide: Macrophage-Expressed Gene 1 protein

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Supramolecule #1: Head-to-head assembly of MPEG-1 (L425K, alpha conformation) solub...

SupramoleculeName: Head-to-head assembly of MPEG-1 (L425K, alpha conformation) soluble pre-pore
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: KSGKPSGEMD EVGVQKCKNA LKLPVLEVLP GGGWDNLRNV DMGRVMELTY SNCRTTEDGQ YIIPDEIFTI PQKQSNLEMN SEILESWANY QSSTSYSINT ELSLFSKVNG KFSTEFQRMK TLQVKDQAIT TRVQVRNLVY TVKINPTLEL SSGFRKELLD ISDRLENNQT ...String:
KSGKPSGEMD EVGVQKCKNA LKLPVLEVLP GGGWDNLRNV DMGRVMELTY SNCRTTEDGQ YIIPDEIFTI PQKQSNLEMN SEILESWANY QSSTSYSINT ELSLFSKVNG KFSTEFQRMK TLQVKDQAIT TRVQVRNLVY TVKINPTLEL SSGFRKELLD ISDRLENNQT RMATYLAELL VLNYGTHVTT SVDAGAALIQ EDHLRASFLQ DSQSSRSAVT ASAGLAFQNT VNFKFEENYT SQNVLTKSYL SNRTNSRVQS IGGVPFYPGI TLQAWQQGIT NHLVAIDRSG LPLHFFINPN MLPDLPGPLV KKVSKTVETA VKRYYTFNTY PGCTDLNSPN FNFQANTDDG SCEGKMTNFS FGGVYQECTQ LSGNRDVLLC QKLEQKNPLT GDFSCPSGYS PVHLLSQIHE EGYNHLECHR KCTLKVFCKT VCEDVFQVAK AEFRAFWCVA SSQVPENSGL LFGGLFSSKS INPMTNAQSC PAGYFPLRLF ENLKVCVSQD YELGSRFAVP FGGFFSCTVG NPLVDPAISR DLGAPSLKKC PGGFSQHPAL ISDGCQVSYC VKSGLFTGGS LPPARLPPFT RPPLMSQAAT NTVIVTNSEN ARSWIKDSQT HQWRLGEPIE LRRAMNVIHG DGGGLSHHHH HH

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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

Particle selectionNumber selected: 133631
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D16 (2x16 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 12578
FSC plot (resolution estimation)

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