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- EMDB-3016: Cryo-EM single particle 3D reconstruction of the native conformat... -

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Entry
Database: EMDB / ID: EMD-3016
TitleCryo-EM single particle 3D reconstruction of the native conformation of E. coli alpha-2-macroglobulin (ECAM)
Map dataReconstruction of the native conformation of the E.coli alpha-2-macroglobulin (ECAM)
Sample
  • Sample: Native 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 / 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 / Bacterial macroglobulin domain 6 ...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: 16.0 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-
UpdateAug 12, 2015-
Current statusAug 12, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.73
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.73
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a42
  • Surface level: 2.73
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3016.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the native conformation of the E.coli alpha-2-macroglobulin (ECAM)
Voxel sizeX=Y=Z: 4.5 Å
Density
Contour LevelBy AUTHOR: 2.73 / Movie #1: 2.73
Minimum - Maximum-2.12648892 - 6.36878967
Average (Standard dev.)0.06237672 (±0.65793127)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions707070
Spacing707070
CellA=B=C: 315.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.54.54.5
M x/y/z707070
origin x/y/z0.0000.0000.000
length x/y/z315.000315.000315.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS707070
D min/max/mean-2.1266.3690.062

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Supplemental data

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Sample components

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Entire : Native conformation of the E. coli alpha-2-macroglobulin (ECAM).

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

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Supramolecule #1000: Native conformation of the E. coli alpha-2-macroglobulin (ECAM).

SupramoleculeName: Native 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: The expressed and purified sample contained the sequence GPM-A40 to P1653.
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: Blotted for 1min before plunging with 5ul purified sample.

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Electron microscopy

MicroscopeOTHER
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 84269 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateJun 10, 2014
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 130

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Image processing

CTF correctionDetails: Each image
Final two d classificationNumber classes: 64
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 16.0 Å / Resolution method: OTHER / Software - Name: XMIPP3, EMAN2 / Number images used: 46842
DetailsThe XMIPP automatic picking routine was used to select particles.

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Chimera
DetailsAn homology model based on the experimental coordinates of native ECAM fragments (PDB codes: 4ZJG, 4ZIU, 4ZJH) induced iECAM (PDB code: 4ZIQ) and the coordinates of native Salmonella enterica serovar typhimurium alpha-2-macroglobulin (PDB code: 4U48) was fitted as a rigid body into the density. Several rounds of manual flexible fitting for MG0-NIE domains were performed to improve the fitting.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: CC
Output model

PDB-5a42:
Cryo-EM single particle 3D reconstruction of the native conformation of E. coli alpha-2-macroglobulin (ECAM)

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: Chimera
DetailsAn homology model based on the experimental coordinates of native ECAM fragments (PDB codes: 4ZJG, 4ZIU, 4ZJH) induced iECAM (PDB code: 4ZIQ) and the coordinates of native Salmonella enterica serovar typhimurium alpha-2-macroglobulin (PDB code: 4U48) was fitted as a rigid body into the density. Several rounds of manual flexible fitting for MG0-NIE domains were performed to improve the fitting.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: CC
Output model

PDB-5a42:
Cryo-EM single particle 3D reconstruction of the native conformation of E. coli alpha-2-macroglobulin (ECAM)

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Atomic model buiding 3

Initial modelPDB ID:
SoftwareName: Chimera
DetailsAn homology model based on the experimental coordinates of native ECAM fragments (PDB codes: 4ZJG, 4ZIU, 4ZJH) induced iECAM (PDB code: 4ZIQ) and the coordinates of native Salmonella enterica serovar typhimurium alpha-2-macroglobulin (PDB code: 4U48) was fitted as a rigid body into the density. Several rounds of manual flexible fitting for MG0-NIE domains were performed to improve the fitting.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: CC
Output model

PDB-5a42:
Cryo-EM single particle 3D reconstruction of the native conformation of E. coli alpha-2-macroglobulin (ECAM)

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Atomic model buiding 4

Initial modelPDB ID:
SoftwareName: Chimera
DetailsAn homology model based on the experimental coordinates of native ECAM fragments (PDB codes: 4ZJG, 4ZIU, 4ZJH) induced iECAM (PDB code: 4ZIQ) and the coordinates of native Salmonella enterica serovar typhimurium alpha-2-macroglobulin (PDB code: 4U48) was fitted as a rigid body into the density. Several rounds of manual flexible fitting for MG0-NIE domains were performed to improve the fitting.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: CC
Output model

PDB-5a42:
Cryo-EM single particle 3D reconstruction of the native conformation of E. coli alpha-2-macroglobulin (ECAM)

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Atomic model buiding 5

Initial modelPDB ID:
SoftwareName: Chimera
DetailsAn homology model based on the experimental coordinates of native ECAM fragments (PDB codes: 4ZJG, 4ZIU, 4ZJH) induced iECAM (PDB code: 4ZIQ) and the coordinates of native Salmonella enterica serovar typhimurium alpha-2-macroglobulin (PDB code: 4U48) was fitted as a rigid body into the density. Several rounds of manual flexible fitting for MG0-NIE domains were performed to improve the fitting.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: CC
Output model

PDB-5a42:
Cryo-EM single particle 3D reconstruction of the native conformation of E. coli alpha-2-macroglobulin (ECAM)

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