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- EMDB-33612: CryoEM structure of QacA (D411N), an antibacterial efflux transpo... -

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Basic information

Entry
Database: EMDB / ID: EMD-33612
TitleCryoEM structure of QacA (D411N), an antibacterial efflux transporter from Staphylococcus aureus
Map dataRefined map obtained from Non-uniform refinement and used for density modification and model building of QacA structure
Sample
  • Complex: Heterotrimer of QacA (54kDa) in complexed with two single domain camelid antibodies (A4/B7)
    • Complex: QacA (54kDa)
      • Protein or peptide: Antiseptic resistance protein
    • Complex: antibodies (A4/B7)
      • Protein or peptide: Single-domain Indian camelid antibody (A4)
      • Protein or peptide: single-domain indian camelid antibody(B7)
KeywordsDrug proton antiporter / major facilitator superfamily(MFS) / Staphylococcus aureus / antibacterial efflux / QacA / MEMBRANE PROTEIN / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


transmembrane transporter activity / plasma membrane
Similarity search - Function
Sugar transport proteins signature 1. / Sugar transporter, conserved site / Major facilitator superfamily / Major Facilitator Superfamily / Major facilitator superfamily domain / Major facilitator superfamily (MFS) profile. / MFS transporter superfamily
Similarity search - Domain/homology
Antiseptic resistance protein
Similarity search - Component
Biological speciesStaphylococcus aureus (bacteria) / Camelus dromedarius (Arabian camel)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsPenmatsa A / Majumder P
Funding support India, 3 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/I/15/2/502063 India
Department of Biotechnology (DBT, India)IA/S/22/1/506242 India
Department of Biotechnology (DBT, India)BT/PR31976/Med/29/1421/2019 India
CitationJournal: EMBO J / Year: 2023
Title: Cryo-EM structure of antibacterial efflux transporter QacA from Staphylococcus aureus reveals a novel extracellular loop with allosteric role.
Authors: Puja Majumder / Shahbaz Ahmed / Pragya Ahuja / Arunabh Athreya / Rakesh Ranjan / Aravind Penmatsa /
Abstract: Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram-positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major ...Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram-positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane helix containing major facilitator superfamily antiporter, mediates proton-coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryo-EM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate-free QacA, complexed to two single-domain camelid antibodies, was elucidated to a resolution of 3.6 Å. The structure displays an outward-open conformation with an extracellular helical hairpin loop (EL7) between transmembrane helices 13 and 14, which is conserved in a subset of DHA2 transporters. Removal of the EL7 hairpin loop or disrupting the interface formed between EL7 and EL1 compromises efflux activity. Chimeric constructs of QacA with a helical hairpin and EL1 grafted from other DHA2 members, LfrA and SmvA, restore activity in the EL7 deleted QacA revealing the allosteric and vital role of EL7 hairpin in antibacterial efflux in QacA and related members.
History
DepositionJun 16, 2022-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33612.png

Downloads & links

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Map

FileDownload / File: emd_33612.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined map obtained from Non-uniform refinement and used for density modification and model building of QacA structure
Voxel sizeX=Y=Z: 0.831 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.041989 - 1.3450524
Average (Standard dev.)0.00029654292 (±0.023213668)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 299.16 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: density modified map for building QacA structure

Fileemd_33612_additional_1.map
Annotationdensity modified map for building QacA structure
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap B flipped for handedness

Fileemd_33612_half_map_1.map
AnnotationHalfmap B flipped for handedness
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A flipped for handed ness

Fileemd_33612_half_map_2.map
AnnotationHalf map A flipped for handed ness
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Heterotrimer of QacA (54kDa) in complexed with two single domain ...

EntireName: Heterotrimer of QacA (54kDa) in complexed with two single domain camelid antibodies (A4/B7)
Components
  • Complex: Heterotrimer of QacA (54kDa) in complexed with two single domain camelid antibodies (A4/B7)
    • Complex: QacA (54kDa)
      • Protein or peptide: Antiseptic resistance protein
    • Complex: antibodies (A4/B7)
      • Protein or peptide: Single-domain Indian camelid antibody (A4)
      • Protein or peptide: single-domain indian camelid antibody(B7)

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Supramolecule #1: Heterotrimer of QacA (54kDa) in complexed with two single domain ...

SupramoleculeName: Heterotrimer of QacA (54kDa) in complexed with two single domain camelid antibodies (A4/B7)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Staphylococcus aureus (bacteria) / Location in cell: membrane
Molecular weightTheoretical: 54 kDa/nm

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Supramolecule #2: QacA (54kDa)

SupramoleculeName: QacA (54kDa) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Camelus dromedarius (Arabian camel)

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Supramolecule #3: antibodies (A4/B7)

SupramoleculeName: antibodies (A4/B7) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3

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Macromolecule #1: Antiseptic resistance protein

MacromoleculeName: Antiseptic resistance protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus aureus (bacteria)
Molecular weightTheoretical: 55.053457 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MISFFTKTTD MMTSKKRWTA LVVLAVSLFV VTMDMTILIM ALPELVRELE PSGTQQLWIV DIYSLVLAGF IIPLSAFADK WGRKKALLT GFALFGLVSL AIFFAESAEF VIAIRFLLGI AGALIMPTTL SMIRVIFENP KERATALAVW SIASSIGAVF G PIIGGALL ...String:
MISFFTKTTD MMTSKKRWTA LVVLAVSLFV VTMDMTILIM ALPELVRELE PSGTQQLWIV DIYSLVLAGF IIPLSAFADK WGRKKALLT GFALFGLVSL AIFFAESAEF VIAIRFLLGI AGALIMPTTL SMIRVIFENP KERATALAVW SIASSIGAVF G PIIGGALL EQFSWHSAFL INVPFAIIAV VAGLFLLPES KLSKEKSHSW DIPSTILSIA GMIGLVWSIK EFSKEGLADI IP WVVIVLA ITMIVIFVKR NLSSSDPMLD VRLFKKRSFS AGTIAAFMTM FAMASVLLLA SQWLQVVEEL SPFKAGLYLL PMA IGDMVF APIAPGLAAR FGPKIVLPSG IGIAAIGMFI MYFFGHPLSY STMALALILV GAGMASLAVA SALIMLETPT SKAG NAAAV EESMYNLGNV FGVAVLGSLS SMLYRVFLDI SSFSSKGIVG DLAHVAEESV VGAVEVAKAT GIKQLANEAV TSFND AFVA TALVGGIIMI IISIVVYLLI PKSLDITKQK

UniProtKB: Antiseptic resistance protein

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Macromolecule #2: Single-domain Indian camelid antibody (A4)

MacromoleculeName: Single-domain Indian camelid antibody (A4) / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Camelus dromedarius (Arabian camel)
Molecular weightTheoretical: 13.697031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQVQLQESGG GSVQTGGSLR LSCAASGYRY SDNCVGWFRQ APGREREAVA TYNSGSSTWY ADSVKGRFTI SQDSAKSTVY LQMNNLKPE DTAMYYCAGR NRLGSYCYMT GDFAYWGQGT QVTVSS

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Macromolecule #3: single-domain indian camelid antibody(B7)

MacromoleculeName: single-domain indian camelid antibody(B7) / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Camelus dromedarius (Arabian camel)
Molecular weightTheoretical: 13.458037 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MQVQLQESGG GSVQAGGSLR LSCAASGYTN SRKCMGWFRQ IPGKEREGVA AIYGFGRGLI LYADSVKGRF TISQDNAKNT VYLQMNSLK PEDTAMYYCA ADSPGSCLSR SGYNYWGQGT QVTVSS

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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

Concentration3 mg/mL
BufferpH: 7
Details: 30mM Hepes, pH7.0 120mM NaCl 2 % glycerol 1mM Undecyl maltoside
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV / Details: blot time of 5 to 6 seconds before plunging.
DetailsQacA complex concentrated to 3 to 4 mg/ml before grid preparation

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Specialist opticsPhase plate: OTHER / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 7770 / Number real images: 7770 / Average exposure time: 2.0 sec. / Average electron dose: 51.0 e/Å2
Details: Images were collected in movie mode at 50 images per movie
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 502364
Startup modelType of model: OTHER / Details: Alphafold2 model of QacA
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 97 / Avg.num./class: 2250
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 97 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF
Details: maps were derived from non uniform refinement follwed by density modification in phenix which yielded a resolution of 3.6 angstroms
Number images used: 218040
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsReal space refinement
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 201
Output model

PDB-7y58:
CryoEM structure of QacA (D411N), an antibacterial efflux transporter from Staphylococcus aureus

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