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- PDB-9c3f: Cryo-EM structure of E. coli AmpG -

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Basic information

Entry
Database: PDB / ID: 9c3f
TitleCryo-EM structure of E. coli AmpG
ComponentsAnhydromuropeptide permease,Soluble cytochrome b562
KeywordsMEMBRANE PROTEIN / Major Facilitator Family transporter / cell wall / antibiotic resistance
Function / homology
Function and homology information


peptidoglycan transmembrane transporter activity / peptidoglycan turnover / symporter activity / electron transport chain / cell wall organization / periplasmic space / electron transfer activity / iron ion binding / heme binding / plasma membrane
Similarity search - Function
AmpG-like permease/Acetyl-coenzyme A transporter 1 / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / Cytochrome b562 / Cytochrome b562 / MFS transporter superfamily / Cytochrome c/b562
Similarity search - Domain/homology
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / Soluble cytochrome b562 / Anhydromuropeptide permease
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsSverak, H. / Worrall, L.J. / Strynadka, N.C.J.
Funding support Canada, 1items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
CitationJournal: Nat Commun / Year: 2024
Title: Cryo-EM characterization of the anydromuropeptide permease AmpG central to bacterial fitness and β-lactam antibiotic resistance.
Authors: Helena E Sverak / Luke N Yaeger / Liam J Worrall / Condurache M Vacariu / Amy J Glenwright / Marija Vuckovic / Zayni-Dean Al Azawi / Ryan P Lamers / Victoria A Marko / Clarissa Skorupski / ...Authors: Helena E Sverak / Luke N Yaeger / Liam J Worrall / Condurache M Vacariu / Amy J Glenwright / Marija Vuckovic / Zayni-Dean Al Azawi / Ryan P Lamers / Victoria A Marko / Clarissa Skorupski / Arvind S Soni / Martin E Tanner / Lori L Burrows / Natalie Cj Strynadka /
Abstract: Bacteria invest significant resources into the continuous creation and tailoring of their essential protective peptidoglycan (PG) cell wall. Several soluble PG biosynthesis products in the periplasm ...Bacteria invest significant resources into the continuous creation and tailoring of their essential protective peptidoglycan (PG) cell wall. Several soluble PG biosynthesis products in the periplasm are transported to the cytosol for recycling, leading to enhanced bacterial fitness. GlcNAc-1,6-anhydroMurNAc and peptide variants are transported by the essential major facilitator superfamily importer AmpG in Gram-negative pathogens including Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa. Accumulation of GlcNAc-1,6-anhydroMurNAc-pentapeptides also results from β-lactam antibiotic induced cell wall damage. In some species, these products upregulate the β-lactamase AmpC, which hydrolyzes β-lactams to allow for bacterial survival and drug-resistant infections. Here, we have used cryo-electron microscopy and chemical synthesis of substrates in an integrated structural, biochemical, and cellular analysis of AmpG. We show how AmpG accommodates the large GlcNAc-1,6-anhydroMurNAc peptides, including a unique hydrophobic vestibule to the substrate binding cavity, and characterize residues involved in binding that inform the mechanism of proton-mediated transport.
History
DepositionMay 31, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 18, 2024Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 18, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jun 4, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jun 4, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Anhydromuropeptide permease,Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,5823
Polymers65,3271
Non-polymers1,2552
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Anhydromuropeptide permease,Soluble cytochrome b562


Mass: 65327.230 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: AmpG with C-terminal BRIL insertion. N-terminal recombinant His-tag cleaved off.
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ampG, b0433, JW0423, cybC / Production host: Escherichia coli (E. coli) / References: UniProt: P0AE16, UniProt: P0ABE7
#2: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H46O11 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM
#3: Chemical ChemComp-PEE / 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine / DOPE


Mass: 744.034 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C41H78NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: DOPE, phospholipid*YM
Has ligand of interestY
Has protein modificationN

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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: AmpG-BRIL-BAG2 / Type: COMPLEX / Details: Complex between AmpG-BRIL and BAG2 antibody / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 500000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0213894
ELECTRON MICROSCOPYf_angle_d1.7135298
ELECTRON MICROSCOPYf_dihedral_angle_d6.561594
ELECTRON MICROSCOPYf_chiral_restr0.038629
ELECTRON MICROSCOPYf_plane_restr0.004634

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