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- EMDB-23932: Cryo-EM structure of the yeast cadmium factor 1 protein (Ycf1p) -

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Basic information

Entry
Database: EMDB / ID: EMD-23932
TitleCryo-EM structure of the yeast cadmium factor 1 protein (Ycf1p)
Map dataEndogenously expressed Ycf1p. Non-uniform refinement.
Sample
  • Organelle or cellular component: Yeast cadmium factor 1 protein (Ycf1p)
    • Protein or peptide: Metal resistance protein YCF1
KeywordsATP binding cassette (ABC) transporter / transmembrane domain 0 (TMD0) / regulatory (R) region / TRANSPORT PROTEIN
Function / homology
Function and homology information


ABC-type Cd2+ transporter / ABC-type cadmium transporter activity / Recycling of bile acids and salts / Heme degradation / Aspirin ADME / Paracetamol ADME / Atorvastatin ADME / P-type cadmium transporter activity / bilirubin transmembrane transporter activity / bilirubin transport ...ABC-type Cd2+ transporter / ABC-type cadmium transporter activity / Recycling of bile acids and salts / Heme degradation / Aspirin ADME / Paracetamol ADME / Atorvastatin ADME / P-type cadmium transporter activity / bilirubin transmembrane transporter activity / bilirubin transport / ABC-family proteins mediated transport / vacuole fusion, non-autophagic / ABC-type glutathione-S-conjugate transporter / ABC-type glutathione S-conjugate transporter activity / fungal-type vacuole membrane / response to metal ion / ATPase-coupled transmembrane transporter activity / response to cadmium ion / glutathione metabolic process / cell redox homeostasis / transmembrane transport / ATP hydrolysis activity / ATP binding / membrane
Similarity search - Function
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 ...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
Metal resistance protein YCF1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBickers SC / Benlekbir S / Rubinstein JL / Kanelis V
Funding support Canada, 3 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2015-05372 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2020-05835 Canada
Canadian Institutes of Health Research (CIHR)PJT162186 Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Structure of Ycf1p reveals the transmembrane domain TMD0 and the regulatory region of ABCC transporters.
Authors: Sarah C Bickers / Samir Benlekbir / John L Rubinstein / Voula Kanelis /
Abstract: ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two ...ATP binding cassette (ABC) proteins typically function in active transport of solutes across membranes. The ABC core structure is composed of two transmembrane domains (TMD1 and TMD2) and two cytosolic nucleotide binding domains (NBD1 and NBD2). Some members of the C-subfamily of ABC (ABCC) proteins, including human multidrug resistance proteins (MRPs), also possess an N-terminal transmembrane domain (TMD0) that contains five transmembrane α-helices and is connected to the ABC core by the L0 linker. While TMD0 was resolved in SUR1, the atypical ABCC protein that is part of the hetero-octameric ATP-sensitive K channel, little is known about the structure of TMD0 in monomeric ABC transporters. Here, we present the structure of yeast cadmium factor 1 protein (Ycf1p), a homolog of human MRP1, determined by electron cryo-microscopy (cryo-EM). A comparison of Ycf1p, SUR1, and a structure of MRP1 that showed TMD0 at low resolution demonstrates that TMD0 can adopt different orientations relative to the ABC core, including a ∼145° rotation between Ycf1p and SUR1. The cryo-EM map also reveals that segments of the regulatory (R) region, which links NBD1 to TMD2 and was poorly resolved in earlier ABCC structures, interacts with the L0 linker, NBD1, and TMD2. These interactions, combined with fluorescence quenching experiments of isolated NBD1 with and without the R region, suggest how posttranslational modifications of the R region modulate ABC protein activity. Mapping known mutations from MRP2 and MRP6 onto the Ycf1p structure explains how mutations involving TMD0 and the R region of these proteins lead to disease.
History
DepositionMay 4, 2021-
Header (metadata) releaseJun 2, 2021-
Map releaseJun 2, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.879
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.879
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7mpe
  • Surface level: 0.879
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23932.png

Downloads & links

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Map

FileDownload / File: emd_23932.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEndogenously expressed Ycf1p. Non-uniform refinement.
Voxel sizeX=Y=Z: 1.03 Å
Density
Contour LevelBy AUTHOR: 0.879 / Movie #1: 0.879
Minimum - Maximum-2.775852 - 4.508105
Average (Standard dev.)0.009326841 (±0.11287288)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 263.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.031.031.03
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z263.680263.680263.680
α/β/γ90.00090.00090.000
start NX/NY/NZ1219875
NX/NY/NZ141223232
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-2.7764.5080.009

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Supplemental data

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Sample components

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Entire : Yeast cadmium factor 1 protein (Ycf1p)

EntireName: Yeast cadmium factor 1 protein (Ycf1p)
Components
  • Organelle or cellular component: Yeast cadmium factor 1 protein (Ycf1p)
    • Protein or peptide: Metal resistance protein YCF1

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Supramolecule #1: Yeast cadmium factor 1 protein (Ycf1p)

SupramoleculeName: Yeast cadmium factor 1 protein (Ycf1p) / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Endogenously expressed Ycf1p that also contains a C-terminal 3x FLAG tag.
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ2168 / Location in cell: Vacoular Membrane
Molecular weightTheoretical: 171.136 KDa

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Macromolecule #1: Metal resistance protein YCF1

MacromoleculeName: Metal resistance protein YCF1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ABC-type Cd2+ transporter
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ2168
Molecular weightTheoretical: 174.024141 KDa
SequenceString: MAGNLVSWAC KLCRSPEGFG PISFYGDFTQ CFIDGVILNL SAIFMITFGI RDLVNLCKKK HSGIKYRRNW IIVSRMALVL LEIAFVSLA SLNISKEEAE NFTIVSQYAS TMLSLFVALA LHWIEYDRSV VANTVLLFYW LFETFGNFAK LINILIRHTY E GIWYSGQT ...String:
MAGNLVSWAC KLCRSPEGFG PISFYGDFTQ CFIDGVILNL SAIFMITFGI RDLVNLCKKK HSGIKYRRNW IIVSRMALVL LEIAFVSLA SLNISKEEAE NFTIVSQYAS TMLSLFVALA LHWIEYDRSV VANTVLLFYW LFETFGNFAK LINILIRHTY E GIWYSGQT GFILTLFQVI TCASILLLEA LPKKPLMPHQ HIHQTLTRRK PNPYDSANIF SRITFSWMSG LMKTGYEKYL VE ADLYKLP RNFSSEELSQ KLEKNWENEL KQKSNPSLSW AICRTFGSKM LLAAFFKAIH DVLAFTQPQL LRILIKFVTD YNS ERQDDH SSLQGFENNH PQKLPIVRGF LIAFAMFLVG FTQTSVLHQY FLNVFNTGMY IKSALTALIY QKSLVLSNEA SGLS STGDI VNLMSVDVQK LQDLTQWLNL IWSGPFQIII CLYSLYKLLG NSMWVGVIIL VIMMPLNSFL MRIQKKLQKS QMKYK DERT RVISEILNNI KSLKLYAWEK PYREKLEEVR NNKELKNLTK LGCYMAVTSF QFNIVPFLVS CCTFAVFVYT EDRALT TDL VFPALTLFNL LSFPLMIIPM VLNSFIEASV SIGRLFTFFT NEELQPDSVQ RLPKVKNIGD VAINIGDDAT FLWQRKP EY KVALKNINFQ AKKGNLTCIV GKVGSGKTAL LSCMLGDLFR VKGFATVHGS VAYVSQVPWI MNGTVKENIL FGHRYDAE F YEKTIKACAL TIDLAILMDG DKTLVGEKGI SLSGGQKARL SLARAVYARA DTYLLDDPLA AVDEHVARHL IEHVLGPNG LLHTKTKVLA TNKVSALSIA DSIALLDNGE ITQQGTYDEI TKDADSPLWK LLNNYGKKNN GKSNEFGDSS ESSVRESSIP VEGELEQLQ KLNDLDFGNS DAISLRRASD ATLGSIDFGD DENIAKREHR EQGKVKWNIY LEYAKACNPK SVCVFILFIV I SMFLSVMG NVWLKHWSEV NSRYGSNPNA ARYLAIYFAL GIGSALATLI QTIVLWVFCT IHASKYLHNL MTNSVLRAPM TF FETTPIG RILNRFSNDI YKVDALLGRT FSQFFVNAVK VTFTITVICA TTWQFIFIII PLSVFYIYYQ QYYLRTSREL RRL DSITRS PIYSHFQETL GGLATVRGYS QQKRFSHINQ CRIDNNMSAF YPSINANRWL AYRLELIGSI IILGAATLSV FRLK QGTLT AGMVGLSLSY ALQITQTLNW IVRMTVEVET NIVSVERIKE YADLKSEAPL IVEGHRPPKE WPSQGDIKFN NYSTR YRPE LDLVLKHINI HIKPNEKVGI VGRTGAGKSS LTLALFRMIE ASEGNIVIDN IAINEIGLYD LRHKLSIIPQ DSQVFE GTV RENIDPINQY TDEAIWRALE LSHLKEHVLS MSNDGLDAQL TEGGGNLSVG QRQLLCLARA MLVPSKILVL DEATAAV DV ETDKVVQETI RTAFKDRTIL TIAHRLNTIM DSDRIIVLDN GKVAEFDSPG QLLSDNKSLF YSLCMEAGLV NENDYKDH D GDYKDHDIDY KDDDDK

UniProtKB: Metal resistance protein YCF1

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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

Concentration5.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTris-HCltris (hydroxymethyl) aminomethane hydrochloride
150.0 mMNaClsodium chloride
0.006 % (w/v)GDNglyco-diosgenin

Details: Buffer was at 4 degrees Celsius.
GridModel: Homemade / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 120 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average exposure time: 9.6 sec. / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2789383
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v.2) / Number images used: 124864
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. v.2)
Final angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. v.2)
Final 3D classificationNumber classes: 1 / Avg.num./class: 124864 / Software - Name: cryoSPARC (ver. v.2)

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