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- EMDB-14049: Cryo-EM structure of ABC transporter STE6-2p from Pichia pastoris... -

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Basic information

Entry
Database: EMDB / ID: EMD-14049
TitleCryo-EM structure of ABC transporter STE6-2p from Pichia pastoris with Verapamil at 3.2 A resolution
Map data
Sample
  • Organelle or cellular component: STE6-2p
    • Protein or peptide: Plasma membrane ATP-binding cassette transporter required for the export of a-factor
  • Ligand: Dexverapamil
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsSchleker ESM / Reinhart C
Funding support Germany, 1 items
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, 2021-
Header (metadata) releaseOct 26, 2022-
Map releaseOct 26, 2022-
UpdateOct 26, 2022-
Current statusOct 26, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14049.png

Downloads & links

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Map

FileDownload / File: emd_14049.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 288 pix.
= 241.056 Å		0.84 Å/pix.
x 288 pix.
= 241.056 Å		0.84 Å/pix.
x 288 pix.
= 241.056 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.837 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.038
Minimum - Maximum-0.1428205 - 0.25909212
Average (Standard dev.)-6.024598e-05 (±0.005785085)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 241.056 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14049_msk_1.map
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Half map: #2

Fileemd_14049_half_map_1.map
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Half map: #1

Fileemd_14049_half_map_2.map
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Sample components

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Entire : STE6-2p

EntireName: STE6-2p
Components
  • Organelle or cellular component: STE6-2p
    • Protein or peptide: Plasma membrane ATP-binding cassette transporter required for the export of a-factor
  • Ligand: Dexverapamil
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en

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Supramolecule #1: STE6-2p

SupramoleculeName: STE6-2p / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Komagataella phaffii CBS 7435 (fungus) / Strain: X-33
Recombinant expressionOrganism: Komagataella phaffii CBS 7435 (fungus) / Recombinant strain: X-33

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Macromolecule #1: Plasma membrane ATP-binding cassette transporter required for the...

MacromoleculeName: Plasma membrane ATP-binding cassette transporter required for the export of a-factor
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Komagataella phaffii CBS 7435 (fungus)
Strain: ATCC 76273 / CBS 7435 / CECT 11047 / NRRL Y-11430 / Wegner 21-1
Molecular weightTheoretical: 144.974266 KDa
Recombinant expressionOrganism: Komagataella phaffii CBS 7435 (fungus)
SequenceString: MVKNYDYKDD DDKGLSFFKR KKYSDKESLS SPETLEEKPN DELDPRVTEI LERQIKADSY GASLVDLYGM LQGWEYCLAV AAYICSIVA GAALPLMTLI FGDMAQQFTD YSSGLHSNNQ FVDKIDENAL YFVYLGVGLL VFNYFATLLH IVVSEIIASR V REKFIWSI ...String:
MVKNYDYKDD DDKGLSFFKR KKYSDKESLS SPETLEEKPN DELDPRVTEI LERQIKADSY GASLVDLYGM LQGWEYCLAV AAYICSIVA GAALPLMTLI FGDMAQQFTD YSSGLHSNNQ FVDKIDENAL YFVYLGVGLL VFNYFATLLH IVVSEIIASR V REKFIWSI LHQNMAYLDS LGSGEITSSI TSDSQLIQQG VSEKIGLAAQ SIATVVSALT VAFVIYWKLA LVLLSVMVAL IL SSTPTIL MLMQAYTDSI ASYGKASSVA EEAFAAIKTA TAFGAHEFQL QKYDEFILES KGYGKKKAIS LALMMGSIWF IVF ATYALA FWQGSRFMVS DNSGIGKILT ACMAMLFGSL IIGNATISLK FVMVGLSAAS KLFAMINREP YFDSASDAGE KINE FDGSI SFRNVTTRYP SRPDITVLSD FTLDIKPGQT IALVGESGSG KSTVIALLER FYEYLDGEIL LDGVDLKSLN IKWVR QQMA LVQQEPVLFA ASIYENVCYG LVGSKYENVT EKVKRELVEK ACKDANAWEF ISQMSNGLDT EVGERGLSLS GGQKQR IAI ARAVISEPKI LLLDEATSAL DTRSEGIVQD ALNRLSETRT TIVIAHRLST IQNADLIVVL SKGKIVETGS HKELLKK KG KYHQLVQIQN IRTKINNSGP QAPISLSNSS DLDSVSHKID RVESLIYERA AADTIDESPV KKQSIPQLFL MLLQINKG D YYLLIPCLFL ALIAGMGFPS FALLAGRVIE AFQVTGPQDF PHMRSLINKY TGFLFMIGCV LLIVYLFLTS FMVLSSESL VYKMRYRCFK QYLRQDMSFF DRPENKVGTL VTTLAKDPQD IEGLSGGTAA QLAVSVVIVV AGIILAVAVN WRLGLVCTAT VPILLGCGF FSVYLLMVFE ERILKDYQES ASYACEQVSA LKTVVSLTRE VGIYEKYSNS IKDQVKRSAR SVSRTTLLYA L IQGMNPWV FALGFWYGSR LLLEGRATNR EFFTVLMAIL FGCQSAGEFF SYAPGMGKAK QAAINIRQVL DTRPKSIDIE SE DGLKIDR LNLKGGIELR DVTFRYPTRP EVPVLTDLNL IIKPGQYVGL VGASGCGKST TVGLIERFYD PESGQVLLDG VDI RDLHLR TYREVLALVQ QEPVLFSGSI RDNIMVGSIS DGADDGSEED MIKACKDANI YDFISSLPEG FDTLCGNKGT MLSG GQKQR VAIARALIRN PRVLLLDEAT SALDSESEMV VQDAIDKASK GRTTITIAHR LSTVQNCDVI YVFDAGRIVE SGKHD ELLQ LRGKYYDLVQ LQGLNAHHHH HHHHHH

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Macromolecule #2: Dexverapamil

MacromoleculeName: Dexverapamil / type: ligand / ID: 2 / Number of copies: 1 / Formula: I6H
Molecular weightTheoretical: 454.602 Da
Chemical component information

ChemComp-I6H:
Dexverapamil / medication, channel blocker*YM / Verapamil

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Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]sp...

MacromoleculeName: (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en
type: ligand / ID: 5 / Number of copies: 1 / Formula: 9Z9
Molecular weightTheoretical: 544.805 Da
Chemical component information

ChemComp-9Z9:
(3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en / detergent*YM

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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

Concentration2.5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 0.0021000000000000003 µm / Nominal defocus min: 0.0011 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 128231
FSC plot (resolution estimation)

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