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- EMDB-41608: NorA single mutant - D307N at pH 7.5 -

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Open data


ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-41608
TitleNorA single mutant - D307N at pH 7.5
Map data
Sample
  • Complex: D307N-NorA:FabDA1
    • Protein or peptide: Quinolone resistance protein NorA
    • Protein or peptide: Heavy Chain of FabDA1 Variable Domain
    • Protein or peptide: Light Chain of FabDA1 Variable Domain
KeywordsD307N NorA / efflux pump / MRSA / TRANSPORT PROTEIN / TRANSPORT PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


xenobiotic transmembrane transporter activity / plasma membrane
Similarity search - Function
Tetracycline resistance protein TetA/multidrug resistance protein MdtG / Multidrug resistance protein / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Quinolone resistance protein NorA
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsLi JP / Li Y / Koide A / Kuang HH / Torres VJ / Koide S / Wang DN / Traaseth NJ
Funding support United States, 9 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI165782 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI108889 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS108151 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK135088 United States
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
National Institutes of Health/Office of the DirectorOD019994 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)RR029300 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM121994 United States
CitationJournal: Nat Commun / Year: 2024
Title: Proton-coupled transport mechanism of the efflux pump NorA.
Authors: Jianping Li / Yan Li / Akiko Koide / Huihui Kuang / Victor J Torres / Shohei Koide / Da-Neng Wang / Nathaniel J Traaseth /
Abstract: Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to ...Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.
History
DepositionAug 13, 2023-
Header (metadata) releaseMay 29, 2024-
Map releaseMay 29, 2024-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41608.png

Downloads & links

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Map

FileDownload / File: emd_41608.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.7842057 - 2.7435107
Average (Standard dev.)0.0017584686 (±0.060021132)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41608_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41608_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41608_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : D307N-NorA:FabDA1

EntireName: D307N-NorA:FabDA1
Components
  • Complex: D307N-NorA:FabDA1
    • Protein or peptide: Quinolone resistance protein NorA
    • Protein or peptide: Heavy Chain of FabDA1 Variable Domain
    • Protein or peptide: Light Chain of FabDA1 Variable Domain

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Supramolecule #1: D307N-NorA:FabDA1

SupramoleculeName: D307N-NorA:FabDA1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 100.656 KDa

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Macromolecule #1: Quinolone resistance protein NorA

MacromoleculeName: Quinolone resistance protein NorA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 46.654863 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNKQIFVLYF NIFLIFLGIG LVIPVLPVYL KDLGLTGSDL GLLVAAFALS QMIISPFGGT LADKLGKKLI ICIGLILFSV SEFMFAVGH NFSVLMLSRV IGGMSAGMVM PGVTGLIADI SPSHQKAKNF GYMSAIINSG FILGPGIGGF MAEVSHRMPF Y FAGALGIL ...String:
MNKQIFVLYF NIFLIFLGIG LVIPVLPVYL KDLGLTGSDL GLLVAAFALS QMIISPFGGT LADKLGKKLI ICIGLILFSV SEFMFAVGH NFSVLMLSRV IGGMSAGMVM PGVTGLIADI SPSHQKAKNF GYMSAIINSG FILGPGIGGF MAEVSHRMPF Y FAGALGIL AFIMSIVLIH DPKKSTTSGF QKLEPQLLTK INWKVFITPV ILTLVLSFGL SAFETLYSLY TADKVNYSPK DI SIAITGG GIFGALFQIY FFDKFMKYFS ELTFIAWSLL YSVVVLILLV FANDYWSIML ISFVVFIGFN MIRPAITNYF SNI AGERQG FAGGLNSTFT SMGNFIGPLI AGALFDVHIE APIYMAIGVS LAGVVIVLIE KQHRAKLKEQ NMENLYFQGK LGPE QKLIS EEDLNSAVDH HHHHHHHHH

UniProtKB: Quinolone resistance protein NorA

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Macromolecule #2: Heavy Chain of FabDA1 Variable Domain

MacromoleculeName: Heavy Chain of FabDA1 Variable Domain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.070568 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
EVQLVESGGG LVQPGGSLRL SCAASGFTFS SSSIHWVRQA PGKGLEWVAS ISSSSGSTSY ADSVKGRFTI SADTSKNTAY LQMNSLRAE DTAVYYCARM SVENHWYYFY WYMSPYAMDY WGQGTLVTV

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Macromolecule #3: Light Chain of FabDA1 Variable Domain

MacromoleculeName: Light Chain of FabDA1 Variable Domain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.209451 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
IQMTQSPSSL SASVGDRVTI TCRASQSVSS AVAWYQQKPG KAPKLLIYSA SSLYSGVPSR FSGSRSGTDF TLTISSLQPE DFATYYCQQ SSSSLITFGQ GTKVEI

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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

BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMTris-HCl
100.0 mMsodilum chlorideNaCl
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Average exposure time: 1.2 sec. / Average electron dose: 52.96 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v.3.3.1) / Number images used: 136284
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v.3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v.3.3.1)
FSC plot (resolution estimation)

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