[English] 日本語
Yorodumi- EMDB-3016: Cryo-EM single particle 3D reconstruction of the native conformat... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3016 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM single particle 3D reconstruction of the native conformation of E. coli alpha-2-macroglobulin (ECAM) | |||||||||
Map data | Reconstruction of the native conformation of the E.coli alpha-2-macroglobulin (ECAM) | |||||||||
Sample |
| |||||||||
Keywords | alpha-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 | |||||||||
Biological species | Escherichia coli K-12 (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 16.0 Å | |||||||||
Authors | Garcia Ferrer I / Arede P / Gomez Blanco J / Luque D / Duquerroy S / Caston JR / Goulas T / Gomis Ruth FX | |||||||||
Citation | Journal: 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3016.map.gz | 1.2 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-3016-v30.xml emd-3016.xml | 13.7 KB 13.7 KB | Display Display | EMDB header |
Images | EMD-3016image500gris.png | 37.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3016 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3016 | HTTPS FTP |
-Validation report
Summary document | emd_3016_validation.pdf.gz | 207.5 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_3016_full_validation.pdf.gz | 206.6 KB | Display | |
Data in XML | emd_3016_validation.xml.gz | 4.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3016 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3016 | HTTPS FTP |
-Related structure data
Related structure data | 5a42MC 3017C 3018C 4ziqC 4ziuC 4zjgC 4zjhC M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_3016.map.gz / Format: CCP4 / Size: 1.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Reconstruction of the native conformation of the E.coli alpha-2-macroglobulin (ECAM) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.5 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : Native conformation of the E. coli alpha-2-macroglobulin (ECAM).
Entire | Name: Native conformation of the E. coli alpha-2-macroglobulin (ECAM). |
---|---|
Components |
|
-Supramolecule #1000: Native conformation of the E. coli alpha-2-macroglobulin (ECAM).
Supramolecule | Name: Native conformation of the E. coli alpha-2-macroglobulin (ECAM). type: sample / ID: 1000 / Oligomeric state: Monomeric / Number unique components: 1 |
---|---|
Molecular weight | Experimental: 180 KDa / Theoretical: 180 KDa / Method: Size exclusion chromatography. |
-Macromolecule #1: alpha-2-macroglobulin
Macromolecule | Name: 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 weight | Experimental: 180 KDa / Theoretical: 180 KDa |
Recombinant expression | Organism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pCRI8b |
Sequence | UniProtKB: Alpha-2-macroglobulin |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.01 mg/mL |
---|---|
Buffer | pH: 7.4 / Details: 10mM Tris-HCL, 75mM NaCl |
Grid | Details: Glow discharged carbon coated Cu/Rh 300 mesh Quantifoil R 1.2/1.3um grids. |
Vitrification | Cryogen name: ETHANE / Instrument: LEICA EM CPC Method: Blotted for 1min before plunging with 5ul purified sample. |
-Electron microscopy
Microscope | OTHER |
---|---|
Date | Jun 10, 2014 |
Image recording | Category: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 130 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 84269 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
-Image processing
Details | The XMIPP automatic picking routine was used to select particles. |
---|---|
CTF correction | Details: Each image |
Final reconstruction | Applied 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 |
Final two d classification | Number classes: 64 |
-Atomic model buiding 1
Initial model | PDB ID: |
---|---|
Software | Name: Chimera |
Details | An 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. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: CC |
Output model | PDB-5a42: |
-Atomic model buiding 2
Initial model | PDB ID: |
---|---|
Software | Name: Chimera |
Details | An 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. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: CC |
Output model | PDB-5a42: |
-Atomic model buiding 3
Initial model | PDB ID: |
---|---|
Software | Name: Chimera |
Details | An 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. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: CC |
Output model | PDB-5a42: |
-Atomic model buiding 4
Initial model | PDB ID: |
---|---|
Software | Name: Chimera |
Details | An 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. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: CC |
Output model | PDB-5a42: |
-Atomic model buiding 5
Initial model | PDB ID: |
---|---|
Software | Name: Chimera |
Details | An 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. |
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Target criteria: CC |
Output model | PDB-5a42: |