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- EMDB-13144: Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward... -

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Basic information

Entry
Database: EMDB / ID: EMD-13144
TitleCryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward-facing conformation with ADP/ATP and rhodamine 6G
Map data
Sample
  • Complex: Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward-facing conformation with ADP/ATP and rhodamine 6G
    • Protein or peptide: Pleiotropic ABC efflux transporter of multiple drugs
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: RHODAMINE 6G
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 (strain ATCC 204508 / S288c) (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsSzewczak-Harris A / Wagner M / Du D / Schmitt L / Luisi BF
Funding support United Kingdom, 1 items
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, 2021-
Header (metadata) releaseNov 10, 2021-
Map releaseNov 10, 2021-
UpdateNov 10, 2021-
Current statusNov 10, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.007213
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.007213
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7p05
  • Surface level: 0.007213
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_13144.png

Downloads & links

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Map

FileDownload / File: emd_13144.map.gz / Format: CCP4 / Size: 548.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.652 Å
Density
Contour LevelBy AUTHOR: 0.007213 / Movie #1: 0.007213
Minimum - Maximum-0.014452262 - 0.033006705
Average (Standard dev.)0.00034423146 (±0.0010624226)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions524524524
Spacing524524524
CellA=B=C: 341.648 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6520.6520.652
M x/y/z524524524
origin x/y/z0.0000.0000.000
length x/y/z341.648341.648341.648
α/β/γ90.00090.00090.000
start NX/NY/NZ7810892
NX/NY/NZ127111124
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS524524524
D min/max/mean-0.0140.0330.000

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Supplemental data

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Sample components

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Entire : Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward...

EntireName: Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward-facing conformation with ADP/ATP and rhodamine 6G
Components
  • Complex: Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward-facing conformation with ADP/ATP and rhodamine 6G
    • Protein or peptide: Pleiotropic ABC efflux transporter of multiple drugs
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: RHODAMINE 6G

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Supramolecule #1: Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward...

SupramoleculeName: Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward-facing conformation with ADP/ATP and rhodamine 6G
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Location in cell: cell membrane

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Macromolecule #1: Pleiotropic ABC efflux transporter of multiple drugs

MacromoleculeName: Pleiotropic ABC efflux transporter of multiple drugs / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 170.61725 KDa
SequenceString: MPEAKLNNNV NDVTSYSSAS SSTENAADLH NYNGFDEHTE ARIQKLARTL TAQSMQNSTQ SAPNKSDAQS IFSSGVEGVN PIFSDPEAP GYDPKLDPNS ENFSSAAWVK NMAHLSAADP DFYKPYSLGC AWKNLSASGA SADVAYQSTV VNIPYKILKS G LRKFQRSK ...String:
MPEAKLNNNV NDVTSYSSAS SSTENAADLH NYNGFDEHTE ARIQKLARTL TAQSMQNSTQ SAPNKSDAQS IFSSGVEGVN PIFSDPEAP GYDPKLDPNS ENFSSAAWVK NMAHLSAADP DFYKPYSLGC AWKNLSASGA SADVAYQSTV VNIPYKILKS G LRKFQRSK ETNTFQILKP MDGCLNPGEL LVVLGRPGSG CTTLLKSISS NTHGFDLGAD TKISYSGYSG DDIKKHFRGE VV YNAEADV HLPHLTVFET LVTVARLKTP QNRIKGVDRE SYANHLAEVA MATYGLSHTR NTKVGNDIVR GVSGGERKRV SIA EVSICG SKFQCWDNAT RGLDSATALE FIRALKTQAD ISNTSATVAI YQCSQDAYDL FNKVCVLDDG YQIYYGPADK AKKY FEDMG YVCPSRQTTA DFLTSVTSPS ERTLNKDMLK KGIHIPQTPK EMNDYWVKSP NYKELMKEVD QRLLNDDEAS REAIK EAHI AKQSKRARPS SPYTVSYMMQ VKYLLIRNMW RLRNNIGFTL FMILGNCSMA LILGSMFFKI MKKGDTSTFY FRGSAM FFA ILFNAFSSLL EIFSLYEARP ITEKHRTYSL YHPSADAFAS VLSEIPSKLI IAVCFNIIFY FLVDFRRNGG VFFFYLL IN IVAVFSMSHL FRCVGSLTKT LSEAMVPASM LLLALSMYTG FAIPKKKILR WSKWIWYINP LAYLFESLLI NEFHGIKF P CAEYVPRGPA YANISSTESV CTVVGAVPGQ DYVLGDDFIR GTYQYYHKDK WRGFGIGMAY VVFFFFVYLF LCEYNEGAK QKGEILVFPR SIVKRMKKRG VLTEKNANDP ENVGERSDLS SDRKMLQESS EEESDTYGEI GLSKSEAIFH WRNLCYEVQI KAETRRILN NVDGWVKPGT LTALMGASGA GKTTLLDCLA ERVTMGVITG DILVNGIPRD KSFPRSIGYC QQQDLHLKTA T VRESLRFS AYLRQPAEVS IEEKNRYVEE VIKILEMEKY ADAVVGVAGE GLNVEQRKRL TIGVELTAKP KLLVFLDEPT SG LDSQTAW SICQLMKKLA NHGQAILCTI HQPSAILMQE FDRLLFMQRG GKTVYFGDLG EGCKTMIDYF ESHGAHKCPA DAN PAEWML EVVGAAPGSH ANQDYYEVWR NSEEYRAVQS ELDWMERELP KKGSITAAED KHEFSQSIIY QTKLVSIRLF QQYW RSPDY LWSKFILTIF NQLFIGFTFF KAGTSLQGLQ NQMLAVFMFT VIFNPILQQY LPSFVQQRDL YEARERPSRT FSWIS FIFA QIFVEVPWNI LAGTIAYFIY YYPIGFYSNA SAAGQLHERG ALFWLFSCAF YVYVGSMGLL VISFNQVAES AANLAS LLF TMSLSFCGVM TTPSAMPRFW IFMYRVSPLT YFIQALLAVG VANVDVKCAD YELLEFTPPS GMTCGQYMEP YLQLAKT GY LTDENATDTC SFCQISTTND YLANVNSFYS ERWRNYGIFI CYIAFNYIAG VFFYWLARVP KKNGKLSKK

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Macromolecule #2: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM / Adenosine diphosphate

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Macromolecule #4: RHODAMINE 6G

MacromoleculeName: RHODAMINE 6G / type: ligand / ID: 4 / Number of copies: 1 / Formula: RHQ
Molecular weightTheoretical: 443.557 Da
Chemical component information

ChemComp-R6G:
RHODAMINE 6G / Rhodamine 6G

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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: 7.8
Component:
ConcentrationFormulaName
50.0 mMTris-HClTris
100.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: HELIUM
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average exposure time: 1.31 sec. / Average electron dose: 47.73 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 76419

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7p05:
Cryo-EM structure of Pdr5 from Saccharomyces cerevisiae in inward-facing conformation with ADP/ATP and rhodamine 6G

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