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- PDB-5a42: Cryo-EM single particle 3D reconstruction of the native conformat... -

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Entry
Database: PDB / ID: 5a42
TitleCryo-EM single particle 3D reconstruction of the native conformation of E. coli alpha-2-macroglobulin (ECAM)
ComponentsUNCHARACTERIZED LIPOPROTEIN YFHM
KeywordsHYDROLASE INHIBITOR / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 16 Å
AuthorsGarcia-Ferrer, I. / Arede, P. / Gomez-Blanco, J. / Luque, D. / Duquerroy, S. / Caston, J.R. / Goulas, T. / Gomis-Ruth, F.X.
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
DepositionJun 4, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 29, 2015Provider: repository / Type: Initial release
Revision 1.1Aug 19, 2015Group: Database references
Revision 1.2Aug 23, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id ..._em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name
Revision 1.3May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: UNCHARACTERIZED LIPOPROTEIN YFHM


Theoretical massNumber of molelcules
Total (without water)177,6901
Polymers177,6901
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein UNCHARACTERIZED LIPOPROTEIN YFHM / ALPHA-2-MACROGLOBULIN


Mass: 177690.297 Da / Num. of mol.: 1 / Fragment: RESIDUES 40-1653
Source method: isolated from a genetically manipulated source
Details: THE EXPRESSED AND PURIFIED SAMPLE CONTAINED THE SEQUENCE GPM-A40 TO P1653.
Source: (gene. exp.) ESCHERICHIA COLI K-12 (bacteria) / Plasmid: PCRI8B / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P76578
Sequence detailsTHE PURIFIED SAMPLE CONTAINED FROM A40 TO P1653

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: NATIVE CONFORMATION OF THE E. COLI ALPHA-2- MACROGLOBULIN (ECAM).
Type: COMPLEX
Buffer solutionName: 10MM TRIS-HCL, 75MM NACL / pH: 7.4 / Details: 10MM TRIS-HCL, 75MM NACL
SpecimenConc.: 0.01 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: CARBON
VitrificationInstrument: LEICA EM CPC / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, INSTRUMENT- LEICA EM CPC, METHOD- BLOTTED FOR 1MIN BEFORE PLUNGING WITH 5UL PURIFIED SAMPLE.,

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 20 / Date: Jun 10, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 84269 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1500 nm
Image recordingFilm or detector model: FEI EAGLE (4k x 4k)
Image scansNum. digital images: 130
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1UCSF Chimeramodel fitting
2EMAN23D reconstruction
3Xmipp33D reconstruction
CTF correctionDetails: EACH IMAGE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: PROJECTION MATCHING / Resolution: 16 Å / Num. of particles: 46842 / Actual pixel size: 4.5 Å
Details: THE FITTED MODEL IS AN HOMOLOGY MODEL BASED ON A ECAM HOMOLOG FROM SALMONELLA ENTERICA (PDB CODE 4U48) AND THE CRYSTAL STRUCTURES ECAM DOMAINS (PDB CODES 4ZIU,4ZJH,4ZJG,4ZIQ). SUBMISSION ...Details: THE FITTED MODEL IS AN HOMOLOGY MODEL BASED ON A ECAM HOMOLOG FROM SALMONELLA ENTERICA (PDB CODE 4U48) AND THE CRYSTAL STRUCTURES ECAM DOMAINS (PDB CODES 4ZIU,4ZJH,4ZJG,4ZIQ). SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3016. (DEPOSITION ID: 13326).
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient / Details: METHOD--RIGID BODY
RefinementHighest resolution: 16 Å
Refinement stepCycle: LAST / Highest resolution: 16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12400 0 0 0 12400

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