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- EMDB-61285: Cryo-EM structure of Outward state Anhydromuropeptide permease (A... -

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Basic information

Entry
Database: EMDB / ID: EMD-61285
TitleCryo-EM structure of Outward state Anhydromuropeptide permease (AmpG) complex with GlcNAc-1,6-anhMurNAc
Map data
Sample
  • Complex: AmpG(G50W/L269W)_BRIL_Fab_nanobody complex
    • Protein or peptide: anti-BRIL Fab Heavy chain
    • Protein or peptide: anti-BRIL Fab Nanobody
    • Protein or peptide: anti-BRIL Fab Light chain
    • Protein or peptide: Muropeptide transporter,Soluble cytochrome b562
  • Ligand: (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
KeywordsAmpG / MFS / Anhydromuropeptide permease / MEMBRANE PROTEIN
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
Muropeptide transporter / Soluble cytochrome b562
Similarity search - Component
Biological speciesYokenella regensburgei (Enteric Group 45) / Homo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsChang N / Kim U / Cho H
Funding support1 items
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, 2024-
Header (metadata) releaseJul 16, 2025-
Map releaseJul 16, 2025-
UpdateJul 16, 2025-
Current statusJul 16, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61285.png

Downloads & links

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Map

FileDownload / File: emd_61285.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.71 Å/pix.
x 500 pix.
= 352.6 Å		0.71 Å/pix.
x 500 pix.
= 352.6 Å		0.71 Å/pix.
x 500 pix.
= 352.6 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.7052 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.9000624 - 1.2793971
Average (Standard dev.)-0.000071146285 (±0.019336157)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 352.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61285_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_61285_half_map_1.map
Projections & Slices
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Half map: #1

Fileemd_61285_half_map_2.map
Projections & Slices
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Sample components

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Entire : AmpG(G50W/L269W)_BRIL_Fab_nanobody complex

EntireName: AmpG(G50W/L269W)_BRIL_Fab_nanobody complex
Components
  • Complex: AmpG(G50W/L269W)_BRIL_Fab_nanobody complex
    • Protein or peptide: anti-BRIL Fab Heavy chain
    • Protein or peptide: anti-BRIL Fab Nanobody
    • Protein or peptide: anti-BRIL Fab Light chain
    • Protein or peptide: Muropeptide transporter,Soluble cytochrome b562
  • Ligand: (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

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Supramolecule #1: AmpG(G50W/L269W)_BRIL_Fab_nanobody complex

SupramoleculeName: AmpG(G50W/L269W)_BRIL_Fab_nanobody complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Yokenella regensburgei (Enteric Group 45)
Molecular weightTheoretical: 124.8 kDa/nm

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Macromolecule #1: anti-BRIL Fab Heavy chain

MacromoleculeName: anti-BRIL Fab Heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.904656 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: EVQLVESGGG LVQPGGSLRL SCAASGFNVV DFSLHWVRQA PGKGLEWVAY ISSSSGSTSY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARW GYWPGEPWWK AFDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ...String:
EVQLVESGGG LVQPGGSLRL SCAASGFNVV DFSLHWVRQA PGKGLEWVAY ISSSSGSTSY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARW GYWPGEPWWK AFDYWGQGTL VTVSSASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKKVEPK

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Macromolecule #2: anti-BRIL Fab Nanobody

MacromoleculeName: anti-BRIL Fab Nanobody / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 13.159438 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
QVQLQESGGG LVQPGGSLRL SCAASGRTIS RYAMSWFRQA PGKEREFVAV ARRSGDGAFY ADSVQGRFTV SRDDAKNTVY LQMNSLKPE DTAVYYCAID SDTFYSGSYD YWGQGTQVTV S

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Macromolecule #3: anti-BRIL Fab Light chain

MacromoleculeName: anti-BRIL Fab Light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.20982 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
DIQMTQSPSS LSASVGDRVT ITCRASQSVS SAVAWYQQKP GKAPKLLIYS ASSLYSGVPS RFSGSRSGTD FTLTISSLQP EDFATYYCQ QYLYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNR

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Macromolecule #4: Muropeptide transporter,Soluble cytochrome b562

MacromoleculeName: Muropeptide transporter,Soluble cytochrome b562 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Yokenella regensburgei (Enteric Group 45)
Molecular weightTheoretical: 64.720977 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: YLRIFQQPKS AILLILGFAS GLPLALTSGT LQAWMTVENI DLKTIWFFSL VGQAYVFKFL WSPVMDRYTP PFLGRRRGWL VTTQILLLI AIAAMGFLEP GTQLRWMAAL AVVIAFCSAS QDIVFDAWKT DVLPAEERGT GAAISVLGYR LGMLVSGGLA L WMADKWLG ...String:
YLRIFQQPKS AILLILGFAS GLPLALTSGT LQAWMTVENI DLKTIWFFSL VGQAYVFKFL WSPVMDRYTP PFLGRRRGWL VTTQILLLI AIAAMGFLEP GTQLRWMAAL AVVIAFCSAS QDIVFDAWKT DVLPAEERGT GAAISVLGYR LGMLVSGGLA L WMADKWLG WQGMYWLMAA LLVPCIIATL LAPEPSADLA DNWETLNDNL KVIEKADNAA QVKDALTKMR AAALDAQKAT PP KLEDKSP DSPEMKDFRH GFDILVGQID DALKLANEGK VKEAQAAAEQ LKTTRNAYIQ KYLGVPRTLE QAVVAPLRDF FGR NNAWLI LLLIVLYKLG DAFAMSLTTT FLIRGVGFDA GEVGMVNKTL GWIATIIGAL YGGVLMQRLS LFRALLIFGI LQGV SNAGY WLLSITDKHL MSMAVAVFFE NLCGGMGTAA FVALLMTLCN KSFSATQFAL LSALSAVGRV YVGPIAGWFV EAHGW PTFY LFSVFAAVPG ILLLLICRKT LEYTQQTESF MMRRHFSGAY QFALYLLLLG CLLLALWLIM LALNAIDYTS FSFLAG LLE VAALIAIAGV LLGAILDYLA LRRTEE

UniProtKB: Muropeptide transporter, Soluble cytochrome b562, Muropeptide transporter

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Macromolecule #5: (2R)-2-[[(1R,2S,3R,4R,5R)-4-acetamido-2-[(2S,3R,4R,5S,6R)-3-aceta...

MacromoleculeName: (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
type: ligand / ID: 5 / Number of copies: 1 / Formula: 2YP
Molecular weightTheoretical: 478.448 Da
Chemical component information

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

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration11 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 173837
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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