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- PDB-7ocy: Enterococcus faecalis EfrCD in complex with a nanobody -

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Basic information

Entry
Database: PDB / ID: 7ocy
TitleEnterococcus faecalis EfrCD in complex with a nanobody
Components
  • (ABC transporter ATP-binding protein) x 2
  • NanobodySingle-domain antibody
KeywordsMEMBRANE PROTEIN / ABC transporter / Nanobody / Enterococcus faecalis
Function / homology
Function and homology information


ABC-type transporter activity / membrane => GO:0016020 / ATP binding
Similarity search - Function
Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ABC transporter ATP-binding protein / ABC transporter ATP-binding protein
Similarity search - Component
Biological speciesEnterococcus faecalis (bacteria)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.25 Å
AuthorsEhrenbolger, K. / Hutter, C.A.J. / Meier, G. / Seeger, M.A. / Barandun, J.
Funding support Sweden, Switzerland, 3items
OrganizationGrant numberCountry
Swedish Research Council2019-02011 Sweden
Swiss National Science FoundationPP00P3_144823 Switzerland
Swiss National Science Foundation310030_188817 Switzerland
CitationJournal: Nat Chem Biol / Year: 2023
Title: Deep mutational scan of a drug efflux pump reveals its structure-function landscape.
Authors: Gianmarco Meier / Sujani Thavarasah / Kai Ehrenbolger / Cedric A J Hutter / Lea M Hürlimann / Jonas Barandun / Markus A Seeger /
Abstract: Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational ...Drug efflux is a common resistance mechanism found in bacteria and cancer cells, but studies providing comprehensive functional insights are scarce. In this study, we performed deep mutational scanning (DMS) on the bacterial ABC transporter EfrCD to determine the drug efflux activity profile of more than 1,430 single variants. These systematic measurements revealed that the introduction of negative charges at different locations within the large substrate binding pocket results in strongly increased efflux activity toward positively charged ethidium, whereas additional aromatic residues did not display the same effect. Data analysis in the context of an inward-facing cryogenic electron microscopy structure of EfrCD uncovered a high-affinity binding site, which releases bound drugs through a peristaltic transport mechanism as the transporter transits to its outward-facing conformation. Finally, we identified substitutions resulting in rapid Hoechst influx without affecting the efflux activity for ethidium and daunorubicin. Hence, single mutations can convert EfrCD into a drug-specific ABC importer.
History
DepositionApr 28, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 18, 2022Provider: repository / Type: Initial release
Revision 1.1May 31, 2023Group: Database references / Refinement description
Category: citation / citation_author / pdbx_initial_refinement_model
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 / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ABC transporter ATP-binding protein
B: ABC transporter ATP-binding protein
C: Nanobody


Theoretical massNumber of molelcules
Total (without water)141,8373
Polymers141,8373
Non-polymers00
Water0
1


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

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Components

#1: Protein ABC transporter ATP-binding protein / ATP-binding cassette domain-containing protein / Multidrug ABC transporter ATP-binding protein


Mass: 62905.289 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterococcus faecalis (bacteria)
Gene: BHU49_08870, BZG32_03820, CUN08_16895, CYQ48_04235, DRJ71_07650, DVW78_12035, EY666_03975, G7X26_03035, GRB94_03325, GTI65_09725, JAOMBFPH_07340
Production host: Lactococcus lactis (lactic acid bacteria) / References: UniProt: A0A1B4XLV2
#2: Protein ABC transporter ATP-binding protein / ABC transporter / ATP-binding/permease protein / ATP-binding cassette domain-containing protein / ...ABC transporter / ATP-binding/permease protein / ATP-binding cassette domain-containing protein / Multidrug ABC transporter permease


Mass: 66213.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterococcus faecalis (bacteria)
Gene: BZG32_03825, CUN08_16890, CYQ48_04230, DVW78_12040, ELS84_1389, EY666_03970, G5T22_05360, GKQ57_11755, GTI65_09720, GTI81_06315, KUB3007_C05280
Production host: Lactococcus lactis (lactic acid bacteria) / References: UniProt: A0A1B4XLV0
#3: Antibody Nanobody / Single-domain antibody


Mass: 12718.138 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli BL21 (bacteria)

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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
1EfrCD in complex with a nanobodyCOMPLEXall0MULTIPLE SOURCES
2Enterococcus faecalisCOMPLEX#1-#21RECOMBINANT
3nanobodySingle-domain antibodyCOMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Enterococcus faecalis (bacteria)1351
23Vicugna pacos (alpaca)30538
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Lactococcus lactis (lactic acid bacteria)1358
33Escherichia coli BL21 (bacteria)511693
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMTris1
2150 mMNaClSodium chloride1
30.15 %beta-DDM1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 %
Details: blot force of -5, waiting time of 1 second, blot time of 4.5 seconds at 4C

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3300 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 52.1 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 3135
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1crYOLO1.3.5particle selection
2EPUimage acquisition
4RELION3.1CTF correction
5CTFFIND4.1.13CTF correction
8UCSF Chimera1.13.1 (build 41965)model fitting
9Coot0.8.9.2model fitting
10ISOLDE1.2model fitting
12RELION3.1initial Euler assignment
13cryoSPARCv3.2final Euler assignment
15cryoSPARCv3.23D reconstruction
22PHENIX1.14-3260model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 720893
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 254682 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Details: Initial docking in Chimera, local adjustments in COOT
Atomic model buildingPDB-ID: 4Q4H

4q4h

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