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- PDB-7qks: Cryo-EM structure of ABC transporter STE6-2p from Pichia pastoris... -

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Basic information

Entry
Database: PDB / ID: 7qks
TitleCryo-EM structure of ABC transporter STE6-2p from Pichia pastoris in apo conformation at 3.1 A resolution
ComponentsPlasma membrane ATP-binding cassette transporter required for the export of a-factor
KeywordsTRANSPORT PROTEIN / ABC transporter / ABCB / MDR / membrane protein / Pichia pastoris
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-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. ...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-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
Plasma membrane ATP-binding cassette transporter required for the export of a-factor
Similarity search - Component
Biological speciesKomagataella phaffii CBS 7435 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSchleker, E.S.M. / Reinhart, C.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Structural and functional investigation of ABC transporter STE6-2p from reveals unexpected interaction with sterol molecules.
Authors: E Sabine M Schleker / Sabine Buschmann / Hao Xie / Sonja Welsch / Hartmut Michel / Christoph Reinhart /
Abstract: Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy ...Adenosine triphosphate (ATP)-binding cassette (ABC) transporters are multidomain transmembrane proteins, which facilitate the transport of various substances across cell membranes using energy derived from ATP hydrolysis. They are important drug targets since they mediate decreased drug susceptibility during pharmacological treatments. For the methylotrophic yeast , a model organism that is a widely used host for protein expression, the role and function of its ABC transporters is unexplored. In this work, we investigated the ABC-B transporter STE6-2p. Functional investigations revealed that STE6-2p is capable of transporting rhodamines in vivo and is active in the presence of verapamil and triazoles in vitro. A phylogenetic analysis displays homology among multidrug resistance (MDR) transporters from pathogenic fungi to human ABC-B transporters. Further, we present high-resolution single-particle electron cryomicroscopy structures of an ABC transporter from in the apo conformation (3.1 Å) and in complex with verapamil and adenylyl imidodiphosphate (AMP-PNP) (3.2 Å). An unknown density between transmembrane helices 4, 5, and 6 in both structures suggests the presence of a sterol-binding site of unknown function.
History
DepositionDec 18, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 26, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Plasma membrane ATP-binding cassette transporter required for the export of a-factor
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,5192
Polymers144,9741
Non-polymers5451
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area50730 Å2

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Components

#1: Protein Plasma membrane ATP-binding cassette transporter required for the export of a-factor


Mass: 144974.266 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Komagataella phaffii CBS 7435 (fungus)
Strain: ATCC 76273 / CBS 7435 / CECT 11047 / NRRL Y-11430 / Wegner 21-1
Gene: STE6-2, PP7435_Chr1-1574 / Production host: Komagataella phaffii CBS 7435 (fungus) / Strain (production host): X-33 / References: UniProt: F2QQK6
#2: Chemical ChemComp-9Z9 / (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en


Mass: 544.805 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H56O5 / Comment: detergent*YM
Has ligand of interestN

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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: STE6-2p / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Komagataella phaffii CBS 7435 (fungus) / Strain: X33
Source (recombinant)Organism: Komagataella phaffii CBS 7435 (fungus) / Strain: X33
Buffer solutionpH: 8
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.1 nm / Nominal defocus min: 1.1 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 89358 / Symmetry type: POINT

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