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- PDB-7p03: Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward... -

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Basic information

Entry
Database: PDB / ID: 7p03
TitleCryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward-facing conformation without nucleotides
ComponentsPleiotropic ABC efflux transporter of multiple drugs
KeywordsTRANSPORT PROTEIN / ABC transporter / antibiotic resistance / membrane protein / fungal infection
Function / homology
Function and homology information


intracellular monoatomic cation homeostasis / xenobiotic detoxification by transmembrane export across the plasma membrane / ABC-type xenobiotic transporter activity / response to cycloheximide / cell periphery / response to xenobiotic stimulus / response to antibiotic / ATP hydrolysis activity / mitochondrion / ATP binding ...intracellular monoatomic cation homeostasis / xenobiotic detoxification by transmembrane export across the plasma membrane / ABC-type xenobiotic transporter activity / response to cycloheximide / cell periphery / response to xenobiotic stimulus / response to antibiotic / ATP hydrolysis activity / mitochondrion / ATP binding / identical protein binding / plasma membrane
Similarity search - Function
Pleiotropic drug resistance protein PDR/CDR / CDR ABC transporter / ABC-transporter, N-terminal domain / ATP-binding cassette transporter, PDR-like subfamily G, domain 1 / ATP-binding cassette transporter, PDR-like subfamily G, domain 2 / CDR ABC transporter / ABC-transporter N-terminal / ABC-2 type transporter / ABC-2 type transporter / ABC transporter-like, conserved site ...Pleiotropic drug resistance protein PDR/CDR / CDR ABC transporter / ABC-transporter, N-terminal domain / ATP-binding cassette transporter, PDR-like subfamily G, domain 1 / ATP-binding cassette transporter, PDR-like subfamily G, domain 2 / CDR ABC transporter / ABC-transporter N-terminal / ABC-2 type transporter / ABC-2 type transporter / ABC transporter-like, conserved site / ABC transporters family signature. / 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
Pleiotropic ABC efflux transporter of multiple drugs
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsSzewczak-Harris, A. / Wagner, M. / Du, D. / Schmitt, L. / Luisi, B.F.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
European Research Council (ERC)742210 United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structure and efflux mechanism of the yeast pleiotropic drug resistance transporter Pdr5.
Authors: Andrzej Harris / Manuel Wagner / Dijun Du / Stefanie Raschka / Lea-Marie Nentwig / Holger Gohlke / Sander H J Smits / Ben F Luisi / Lutz Schmitt /
Abstract: Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux ...Pdr5, a member of the extensive ABC transporter superfamily, is representative of a clinically relevant subgroup involved in pleiotropic drug resistance. Pdr5 and its homologues drive drug efflux through uncoupled hydrolysis of nucleotides, enabling organisms such as baker's yeast and pathogenic fungi to survive in the presence of chemically diverse antifungal agents. Here, we present the molecular structure of Pdr5 solved with single particle cryo-EM, revealing details of an ATP-driven conformational cycle, which mechanically drives drug translocation through an amphipathic channel, and a clamping switch within a conserved linker loop that acts as a nucleotide sensor. One half of the transporter remains nearly invariant throughout the cycle, while its partner undergoes changes that are transmitted across inter-domain interfaces to support a peristaltic motion of the pumped molecule. The efflux model proposed here rationalises the pleiotropic impact of Pdr5 and opens new avenues for the development of effective antifungal compounds.
History
DepositionJun 29, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 10, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Pleiotropic ABC efflux transporter of multiple drugs


Theoretical massNumber of molelcules
Total (without water)170,6171
Polymers170,6171
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area56390 Å2

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Components

#1: Protein Pleiotropic ABC efflux transporter of multiple drugs / Pleiotropic drug resistance protein 5 / Suppressor of toxicity of sporidesmin


Mass: 170617.250 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / References: UniProt: P33302

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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

ComponentName: Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward-facing conformation without nucleotide
Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Cellular location: cell membrane
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HClTris1
2100 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 magnification: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: HELIUM / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.31 sec. / Electron dose: 48.13 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7Cootmodel fitting
9PHENIX1.18.2model refinement
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
12cryoSPARC3classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.45 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68154 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00610926
ELECTRON MICROSCOPYf_angle_d0.61214809
ELECTRON MICROSCOPYf_dihedral_angle_d13.4781460
ELECTRON MICROSCOPYf_chiral_restr0.0431616
ELECTRON MICROSCOPYf_plane_restr0.0051872

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