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- PDB-8zbb: Cryo-EM structure of outward state Anhydromuropeptide permease (A... -

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Basic information

Entry
Database: PDB / ID: 8zbb
TitleCryo-EM structure of outward state Anhydromuropeptide permease (AmpG) G50W/L269W
Components
  • Muropeptide transporter,Soluble cytochrome b562
  • anti-BRIL Fab Heavy chain
  • anti-BRIL Fab Light chain
  • anti-BRIL Fab Nanobody
KeywordsPEPTIDE BINDING PROTEIN / transporter / ampg / cryo-em / permease
Function / homology
Function and homology information


transmembrane transporter activity / electron transport chain / periplasmic space / electron transfer activity / iron ion binding / heme binding / 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 / Cytochrome c/b562 / MFS transporter superfamily
Similarity search - Domain/homology
Anhydromuropeptide permease / Soluble cytochrome b562
Similarity search - Component
Biological speciesYokenella regensburgei (Enteric Group 45)
Escherichia coli (E. coli)
Homo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsYoo, Y. / Chang, N. / Kim, U. / Kim, H. / 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
DepositionApr 26, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 30, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Apr 30, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 2.0Jun 11, 2025Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Other / Polymer sequence / Refinement description / Source and taxonomy / Structure summary
Category: atom_site / em_admin ...atom_site / em_admin / em_entity_assembly / em_entity_assembly_molwt / em_entity_assembly_naturalsource / em_image_recording / em_imaging / em_single_particle_entity / em_software / entity / entity_name_com / entity_poly / entity_poly_seq / entity_src_gen / pdbx_contact_author / pdbx_entry_details / pdbx_modification_feature / pdbx_poly_seq_scheme / pdbx_struct_assembly / pdbx_struct_assembly_auth_evidence / pdbx_struct_assembly_gen / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_rmsd_angle / pdbx_validate_torsion / refine / refine_ls_restr / space_group / space_group_symop / struct_asym / struct_conf / struct_conn / struct_mon_prot_cis / struct_ref / struct_ref_seq / struct_ref_seq_dif / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _em_admin.last_update / _em_entity_assembly.entity_id_list ..._em_admin.last_update / _em_entity_assembly.entity_id_list / _em_entity_assembly.name / _em_image_recording.avg_electron_dose_per_image / _em_imaging.microscope_model / _em_imaging.nominal_defocus_min / _em_software.category / _em_software.image_processing_id / _em_software.imaging_id / _em_software.name / _em_software.version / _pdbx_entry_details.has_protein_modification / _pdbx_struct_assembly.oligomeric_count / _pdbx_struct_assembly.oligomeric_details / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_validate_rmsd_angle.angle_deviation / _pdbx_validate_rmsd_angle.angle_standard_deviation / _pdbx_validate_rmsd_angle.angle_target_value / _pdbx_validate_rmsd_angle.angle_value / _pdbx_validate_rmsd_angle.auth_atom_id_1 / _pdbx_validate_rmsd_angle.auth_atom_id_2 / _pdbx_validate_rmsd_angle.auth_atom_id_3 / _pdbx_validate_rmsd_angle.auth_comp_id_1 / _pdbx_validate_rmsd_angle.auth_comp_id_2 / _pdbx_validate_rmsd_angle.auth_comp_id_3 / _pdbx_validate_rmsd_angle.auth_seq_id_1 / _pdbx_validate_rmsd_angle.auth_seq_id_2 / _pdbx_validate_rmsd_angle.auth_seq_id_3 / _refine.ls_d_res_high / _refine.pdbx_ls_cross_valid_method / _refine.pdbx_stereochemistry_target_values
Description: Model completeness
Details: An additional chain was modeled into previously unoccupied map density, based on reviewer request, to improve model completeness.
Provider: author / Type: Coordinate replacement
Revision 1.1Jun 11, 2025Data content type: EM metadata
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Group: Data collection / Data processing ...Data collection / Data processing / Database references / Experimental summary / Other / Source and taxonomy / Structure summary
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Category: em_admin / em_entity_assembly ...em_admin / em_entity_assembly / em_entity_assembly_molwt / em_entity_assembly_naturalsource / em_image_recording / em_imaging / em_single_particle_entity / em_software / entity / entity_poly / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Data content type: EM metadata / EM metadata ...EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata / EM metadata
Item: _em_admin.last_update / _em_entity_assembly.entity_id_list ..._em_admin.last_update / _em_entity_assembly.entity_id_list / _em_entity_assembly.name / _em_image_recording.avg_electron_dose_per_image / _em_imaging.microscope_model / _em_imaging.nominal_defocus_min / _em_software.category / _em_software.image_processing_id / _em_software.imaging_id / _em_software.name / _em_software.version
Revision 2.1Jul 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)124,0454
Polymers124,0454
Non-polymers00
Water00
1


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

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Components

#1: Protein Muropeptide transporter,Soluble cytochrome b562 / Cytochrome b-562


Mass: 63770.863 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
#2: 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) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: 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)
#4: 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) / Production host: Escherichia coli BL21(DE3) (bacteria)
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(Apo) / Type: COMPLEX / Entity ID: all / 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: 200 nm
Image recordingElectron dose: 60 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
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 236303 / Symmetry type: POINT
RefinementHighest resolution: 3.11 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0048925
ELECTRON MICROSCOPYf_angle_d0.66112144
ELECTRON MICROSCOPYf_dihedral_angle_d4.7771224
ELECTRON MICROSCOPYf_chiral_restr0.0421386
ELECTRON MICROSCOPYf_plane_restr0.0051523

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