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- PDB-9j9z: Cryo-EM structure of Outward state Anhydromuropeptide permease (A... -

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Basic information

Entry
Database: PDB / ID: 9j9z
TitleCryo-EM structure of Outward state Anhydromuropeptide permease (AmpG) complex with GlcNAc-1,6-anhMurNAc
Components
  • Muropeptide transporter,Soluble cytochrome b562
  • anti-BRIL Fab Heavy chain
  • anti-BRIL Fab Light chain
  • anti-BRIL Fab Nanobody
KeywordsMEMBRANE PROTEIN / AmpG / MFS / Anhydromuropeptide permease
Function / homology
Function and homology information


electron transport chain / periplasmic space / electron transfer activity / iron ion binding / heme binding
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 / Cytochrome c/b562 / MFS transporter superfamily
Similarity search - Domain/homology
Chem-2YP / Muropeptide transporter / Soluble cytochrome b562
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
Yokenella regensburgei (Enteric Group 45)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsChang, N. / Kim, U. / Cho, H.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2025
Title: Structural and functional insights of AmpG in muropeptide transport and multiple β-lactam antibiotics resistance.
Authors: Nienping Chang / Hoyoung Kim / Uijin Kim / Yongju Cho / Youngki Yoo / Hyunsook Lee / Ji Won Kim / Min Sung Kim / Jaeho Lee / Young-Lag Cho / Kitae Kim / Dongeun Yong / Hyun-Soo Cho /
Abstract: Anhydromuropeptide permease (AmpG) is a transporter protein located in the inner membrane of certain gram -negative bacteria, involved in peptidoglycan (PG) recycling and β-lactamase induction. ...Anhydromuropeptide permease (AmpG) is a transporter protein located in the inner membrane of certain gram -negative bacteria, involved in peptidoglycan (PG) recycling and β-lactamase induction. Decreased AmpG function reduces resistance of antibiotic-resistant bacteria to β-lactam antibiotics. Therefore, AmpG-targeting inhibitors are promising 'antibiotic adjuvants'. However, as the tertiary structure of AmpG has not yet been identified, the development of targeted inhibitors remains challenging. We present four cryo-electron microscopy (cryo-EM) structures: the apo-inward and apo-outward state structures and the inward-occluded and outward states complexed with the substrate GlcNAc-1,6-anhMurNAc. Through functional analysis and molecular dynamics (MD) simulations, we identified motif A, which stabilizes the outward state, substrate-binding pocket, and protonation-related residues. Based on the structure of AmpG and our experimental results, we propose a muropeptide transport mechanism for AmpG. A deeper understanding of its structure and transport mechanism provides a foundation for the development of antibiotic adjuvants.
History
DepositionAug 23, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 16, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
H: anti-BRIL Fab Heavy chain
K: anti-BRIL Fab Nanobody
L: anti-BRIL Fab Light chain
A: Muropeptide transporter,Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,4735
Polymers124,9954
Non-polymers4781
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody anti-BRIL Fab Heavy chain


Mass: 23904.656 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IGHG1 / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Antibody anti-BRIL Fab Nanobody


Mass: 13159.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Antibody anti-BRIL Fab Light chain


Mass: 23209.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: IGHG1 / Production host: Escherichia coli BL21(DE3) (bacteria)
#4: Protein Muropeptide transporter,Soluble cytochrome b562 / Cytochrome b-562


Mass: 64720.977 Da / Num. of mol.: 1 / Mutation: G50W/L269W
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Yokenella regensburgei (Enteric Group 45), (gene. exp.) Escherichia coli (E. coli)
Gene: ampG, NCTC11967_01129, cybC / Production host: Escherichia coli (E. coli) / References: UniProt: A0AB38FS76, UniProt: P0ABE7
#5: Chemical ChemComp-2YP / (2R)-2-[[(1R,2S,3R,4R,5R)-4-acetamido-2-[(2S,3R,4R,5S,6R)-3-acetamido-6-(hydroxymethyl)-4,5-bis(oxidanyl)oxan-2-yl]oxy-6,8-dioxabicyclo[3.2.1]octan-3-yl]oxy]propanoic acid


Mass: 478.448 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H30N2O12 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationY

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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(G50W/L269W)_BRIL_Fab_nanobody complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 124.8 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
11Yokenella regensburgei (Enteric Group 45)158877
21Homo sapiens (human)9606
31synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 11 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 173837 / Symmetry type: POINT
RefinementHighest resolution: 2.97 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049030
ELECTRON MICROSCOPYf_angle_d0.71312290
ELECTRON MICROSCOPYf_dihedral_angle_d17.8053216
ELECTRON MICROSCOPYf_chiral_restr0.041406
ELECTRON MICROSCOPYf_plane_restr0.0081539

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