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- EMDB-3018: Cryo-EM single particle 3D reconstruction of the induced, monomer... -

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Basic information

Entry
Database: EMDB / ID: EMD-3018
TitleCryo-EM single particle 3D reconstruction of the induced, monomeric conformation of E. coli alpha-2-macroglobulin (ECAM)
Map dataReconstruction of induced monomeric Escherichia coli alpha-2-macroglobulin
Sample
  • Sample: Induced, monomeric conformation of the E. coli alpha-2-macroglobulin (ECAM).
  • Protein or peptide: alpha-2-macroglobulin
Keywordsalpha-2-macroglobulin / ECAM / peptidase inhibitor
Function / homology
Function and homology information


endopeptidase inhibitor activity / defense response / protein homodimerization activity / extracellular space / plasma membrane
Similarity search - Function
Alpha-2-macroglobulin MG3 domain / Alpha-2-macroglobulin, bacteria / Alpha-2-macroglobulin, MG1 domain / Bacterial Alpha-2-macroglobulin, MG5 domain / Bacterial alpha-2-macroglobulin MG10 domain / Bacterial Alpha-2-macroglobulin, MG6 domain / : / : / : / Bacterial alpha-2-macroglobulin MG3 domain ...Alpha-2-macroglobulin MG3 domain / Alpha-2-macroglobulin, bacteria / Alpha-2-macroglobulin, MG1 domain / Bacterial Alpha-2-macroglobulin, MG5 domain / Bacterial alpha-2-macroglobulin MG10 domain / Bacterial Alpha-2-macroglobulin, MG6 domain / : / : / : / Bacterial alpha-2-macroglobulin MG3 domain / Bacterial macroglobulin domain 6 / Bacterial Alpha-2-macroglobulin MG1 domain / Bacterial Alpha-2-macroglobulin MG5 domain / Bacterial Alpha-2-macroglobulin MG10 domain / Bacterial alpha-2 macroglobulin MG2 domain / A2MG, CUB domain / : / Alpha-macro-globulin thiol-ester bond-forming region / Alpha-2-macroglobulin / Macroglobulin domain / Alpha-2-macroglobulin, bait region domain / Alpha-macroglobulin-like, TED domain / Alpha-2-macroglobulin family / MG2 domain / A-macroglobulin TED domain / Alpha-2-macroglobulin bait region domain / Alpha-2-Macroglobulin / Alpha-2-macroglobulin family / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid / Prokaryotic membrane lipoprotein lipid attachment site profile.
Similarity search - Domain/homology
Alpha-2-macroglobulin
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.0 Å
AuthorsGarcia Ferrer I / Arede P / Gomez Blanco J / Luque D / Duquerroy S / Caston JR / Goulas T / Gomis Ruth FX
CitationJournal: Proc Natl Acad Sci U S A / Year: 2015
Title: Structural and functional insights into Escherichia coli α2-macroglobulin endopeptidase snap-trap inhibition.
Authors: Irene Garcia-Ferrer / Pedro Arêde / Josué Gómez-Blanco / Daniel Luque / Stephane Duquerroy / José R Castón / Theodoros Goulas / F Xavier Gomis-Rüth /
Abstract: The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2- ...The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ∼ 180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region. Structural studies by electron microscopy and crystallography reveal that this cleavage causes major structural rearrangement of more than half the 13-domain structure from a native to a compact induced form. It also exposes a reactive thioester bond, which covalently traps the peptidase. Subsequently, peptidase-laden ECAM is shed from the membrane and may dimerize. Trapped peptidases are still active except against very large substrates, so inhibition potentially prevents damage of large cell envelope components, but not host digestion. Mechanistically, these results document a novel monomeric "snap trap."
History
DepositionMay 15, 2015-
Header (metadata) releaseJun 10, 2015-
Map releaseJun 17, 2015-
UpdateJul 22, 2015-
Current statusJul 22, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.15
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3018imag...

Downloads & links

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Map

FileDownload / File: emd_3018.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of induced monomeric Escherichia coli alpha-2-macroglobulin
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.7 Å/pix.
x 70 pix.
= 259. Å		3.7 Å/pix.
x 70 pix.
= 259. Å		3.7 Å/pix.
x 70 pix.
= 259. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.15 / Movie #1: 1.15
Minimum - Maximum-0.87479591 - 2.37521148
Average (Standard dev.)0.02536137 (±0.27807739)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions707070
Spacing707070
CellA=B=C: 259.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.73.73.7
M x/y/z707070
origin x/y/z0.0000.0000.000
length x/y/z259.000259.000259.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-24-24-24
NX/NY/NZ494949
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS707070
D min/max/mean-0.8752.3750.025

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Supplemental data

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Sample components

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Entire : Induced, monomeric conformation of the E. coli alpha-2-macroglobu...

EntireName: Induced, monomeric conformation of the E. coli alpha-2-macroglobulin (ECAM).
Components
  • Sample: Induced, monomeric conformation of the E. coli alpha-2-macroglobulin (ECAM).
  • Protein or peptide: alpha-2-macroglobulin

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Supramolecule #1000: Induced, monomeric conformation of the E. coli alpha-2-macroglobu...

SupramoleculeName: Induced, monomeric conformation of the E. coli alpha-2-macroglobulin (ECAM).
type: sample / ID: 1000 / Oligomeric state: Monomeric / Number unique components: 1
Molecular weightExperimental: 180 KDa / Theoretical: 180 KDa / Method: Size exclusion chromatography.

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Macromolecule #1: alpha-2-macroglobulin

MacromoleculeName: alpha-2-macroglobulin / type: protein_or_peptide / ID: 1 / Name.synonym: ECAM
Details: ECAM was reacted with trypsin and the resulting induced, monomeric ECAM was purified by size-exclusion chromatography.
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Location in cell: Periplasm
Molecular weightExperimental: 180 KDa / Theoretical: 180 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pCRI8b
SequenceUniProtKB: Alpha-2-macroglobulin

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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

Concentration0.01 mg/mL
BufferpH: 7.4 / Details: 10mM Tris-HCL, 75mM NaCl
GridDetails: Glow discharged carbon coated Cu/Rh 300 mesh Quantifoil R 1.2/1.3um grids.
VitrificationCryogen name: ETHANE / Instrument: LEICA EM CPC
Method: Blot for 1min before plunging with 5ul purified sample.

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Electron microscopy

MicroscopeOTHER
DateJul 17, 2014
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 84
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 84269 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsThe XMIPP automatic picking routine was used to select particles.
CTF correctionDetails: Each image
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: XMIPP3, EMAN2 / Number images used: 18346
Final two d classificationNumber classes: 64

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