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- PDB-7y58: CryoEM structure of QacA (D411N), an antibacterial efflux transpo... -

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Basic information

Entry
Database: PDB / ID: 7y58
TitleCryoEM structure of QacA (D411N), an antibacterial efflux transporter from Staphylococcus aureus
Components
  • Antiseptic resistance protein
  • Single-domain Indian camelid antibody (A4)
  • single-domain indian camelid antibody(B7)
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / Drug 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsPenmatsa, A. / Majumder, P.
Funding support India, 3items
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, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 12, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2023Group: Data collection
Category: chem_comp_atom / chem_comp_bond / pdbx_validate_planes
Item: _pdbx_validate_planes.type
Revision 1.2Nov 22, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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 / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Antiseptic resistance protein
B: Single-domain Indian camelid antibody (A4)
C: single-domain indian camelid antibody(B7)


Theoretical massNumber of molelcules
Total (without water)82,2093
Polymers82,2093
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Antiseptic resistance protein / Efflux protein QacA / Multidrug efflux pump / QacA / Quaternary ammonium compound efflux MFS transporter QacA


Mass: 55053.457 Da / Num. of mol.: 1 / Mutation: D411N
Source method: isolated from a genetically manipulated source
Details: synthetic codon optimized / Source: (gene. exp.) Staphylococcus aureus (bacteria)
Gene: qacA, GZ128_13815, GZ156_13500, SAP062D_001, SAP066A_020, SAP094B_019, SAP098A_005, SAP100B_004, SAP101A_020, SAP104C_021
Production host: Escherichia coli (E. coli) / References: UniProt: Q1XG09
#2: Antibody Single-domain Indian camelid antibody (A4)


Mass: 13697.031 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelus dromedarius (Arabian camel) / Production host: Escherichia coli (E. coli) / Strain (production host): Shuffle T7
#3: Antibody single-domain indian camelid antibody(B7)


Mass: 13458.037 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Camelus dromedarius (Arabian camel) / Production host: Escherichia coli (E. coli) / Strain (production host): Shuffle T7

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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

Component
IDNameTypeEntity IDParent-IDSource
1Heterotrimer of QacA (54kDa) in complexed with two single domain camelid antibodies (A4/B7)COMPLEXall0MULTIPLE SOURCES
2QacA (54kDa)COMPLEX#11RECOMBINANT
3antibodies (A4/B7)COMPLEX#2-#31RECOMBINANT
Molecular weightValue: 54 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular location
21Staphylococcus aureus (bacteria)1280membrane
32Camelus dromedarius (Arabian camel)9838
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
21Escherichia coli (E. coli)562pBAD
32Escherichia coli (E. coli)562
Buffer solutionpH: 7
Details: 30mM Hepes, pH7.0 120mM NaCl 2 % glycerol 1mM Undecyl maltoside
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: QacA complex concentrated to 3 to 4 mg/ml before grid preparation
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K / Details: blot time of 5 to 6 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 4500 nm / Nominal defocus min: 1500 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2 sec. / Electron dose: 51 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 7770 / Num. of real images: 7770
Details: Images were collected in movie mode at 50 images per movie
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV / Phase plate: OTHER

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Processing

EM softwareName: PHENIX / Version: 1.20rc4_4425: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 502364
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 218040
Details: maps were derived from non uniform refinement follwed by density modification in phenix which yielded a resolution of 3.6 angstroms
Num. of class averages: 97 / Symmetry type: POINT
Atomic model buildingB value: 201 / Protocol: AB INITIO MODEL / Space: REAL / Details: Real space refinement
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035724
ELECTRON MICROSCOPYf_angle_d0.7027765
ELECTRON MICROSCOPYf_dihedral_angle_d5.412828
ELECTRON MICROSCOPYf_chiral_restr0.041911
ELECTRON MICROSCOPYf_plane_restr0.005962

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