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- EMDB-23691: E1435Q Ycf1 mutant in inward-facing wide conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-23691
TitleE1435Q Ycf1 mutant in inward-facing wide conformation
Map dataSharpened refine map constructed on unsharpened map using provided mask by Sharpen 3D - cisTEM.
Sample
  • Complex: Ycf1
    • Protein or peptide: Metal resistance protein YCF1
Function / homology
Function and homology information


ABC-type Cd2+ transporter / ABC-type cadmium transporter activity / Recycling of bile acids and salts / Heme degradation / Cytoprotection by HMOX1 / Aspirin ADME / Paracetamol ADME / Atorvastatin ADME / Transport of RCbl within the body / Synthesis of Leukotrienes (LT) and Eoxins (EX) ...ABC-type Cd2+ transporter / ABC-type cadmium transporter activity / Recycling of bile acids and salts / Heme degradation / Cytoprotection by HMOX1 / Aspirin ADME / Paracetamol ADME / Atorvastatin ADME / Transport of RCbl within the body / Synthesis of Leukotrienes (LT) and Eoxins (EX) / 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 S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsKhandelwal NK / Millan CR / Thaker TM / Tomasiak TM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM114245 United States
CitationJournal: Nat Commun / Year: 2022
Title: The structural basis for regulation of the glutathione transporter Ycf1 by regulatory domain phosphorylation.
Authors: Nitesh Kumar Khandelwal / Cinthia R Millan / Samantha I Zangari / Samantha Avila / Dewight Williams / Tarjani M Thaker / Thomas M Tomasiak /
Abstract: Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of ...Yeast Cadmium Factor 1 (Ycf1) sequesters heavy metals and glutathione into the vacuole to counter cell stress. Ycf1 belongs to the ATP binding cassette C-subfamily (ABCC) of transporters, many of which are regulated by phosphorylation on intrinsically-disordered domains. The regulatory mechanism of phosphorylation is still poorly understood. Here, we report two cryo-EM structures of Ycf1 at 3.4 Å and 4.0 Å resolution in inward-facing open conformations that capture previously unobserved ordered states of the intrinsically disordered regulatory domain (R-domain). R-domain phosphorylation is clearly evident and induces a topology promoting electrostatic and hydrophobic interactions with Nucleotide Binding Domain 1 (NBD1) and the Lasso motif. These interactions stay constant between the structures and are related by rigid body movements of the NBD1/R-domain complex. Biochemical data further show R-domain phosphorylation reorganizes the Ycf1 architecture and is required for maximal ATPase activity. Together, we provide insights into how R-domains control ABCC transporter activity.
History
DepositionMar 25, 2021-
Header (metadata) releaseApr 6, 2022-
Map releaseApr 6, 2022-
UpdateOct 19, 2022-
Current statusOct 19, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_23691.png

Downloads & links

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Map

FileDownload / File: emd_23691.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened refine map constructed on unsharpened map using provided mask by Sharpen 3D - cisTEM.
Voxel sizeX=Y=Z: 1.031 Å
Density
Contour LevelBy AUTHOR: 3.5
Minimum - Maximum-11.312036 - 16.05509
Average (Standard dev.)-0.00015694681 (±0.758478)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 309.30002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_23691_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map obtained from Manual Refine - cisTEM.

Fileemd_23691_additional_1.map
AnnotationUnsharpened map obtained from Manual Refine - cisTEM.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: The second half-map from unsharpened refine map generated...

Fileemd_23691_half_map_1.map
AnnotationThe second half-map from unsharpened refine map generated in Generate 3D - cisTEM.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: The first half-map from unsharpened refine map generated...

Fileemd_23691_half_map_2.map
AnnotationThe first half-map from unsharpened refine map generated in Generate 3D - cisTEM.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ycf1

EntireName: Ycf1
Components
  • Complex: Ycf1
    • Protein or peptide: Metal resistance protein YCF1

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Supramolecule #1: Ycf1

SupramoleculeName: Ycf1 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant strain: DSY-5
Molecular weightTheoretical: 176.66831 KDa

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Macromolecule #1: Metal resistance protein YCF1

MacromoleculeName: Metal resistance protein YCF1 / type: protein_or_peptide / ID: 1
Details: Phosphorylated residues (S908, T911 and S914) are present in the structure file.
Number of copies: 1 / Enantiomer: LEVO / EC number: ABC-type Cd2+ transporter
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c
Molecular weightTheoretical: 176.318328 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: ASDYKDDDDK GALEVLFQGP SSPMAGNLVS WACKLCRSPE GFGPISFYGD FTQCFIDGVI LNLSAIFMIT FGIRDLVNLC KKKHSGIKY RRNWIIVSRM ALVLLEIAFV SLASLNISKE EAENFTIVSQ YASTMLSLFV ALALHWIEYD RSVVANTVLL F YWLFETFG ...String:
ASDYKDDDDK GALEVLFQGP SSPMAGNLVS WACKLCRSPE GFGPISFYGD FTQCFIDGVI LNLSAIFMIT FGIRDLVNLC KKKHSGIKY RRNWIIVSRM ALVLLEIAFV SLASLNISKE EAENFTIVSQ YASTMLSLFV ALALHWIEYD RSVVANTVLL F YWLFETFG NFAKLINILI RHTYEGIWYS GQTGFILTLF QVITCASILL LEALPKKPLM PHQHIHQTLT RRKPNPYDSA NI FSRITFS WMSGLMKTGY EKYLVEADLY KLPRNFSSEE LSQKLEKNWE NELKQKSNPS LSWAICRTFG SKMLLAAFFK AIH DVLAFT QPQLLRILIK FVTDYNSERQ DDHSSLQGFE NNHPQKLPIV RGFLIAFAMF LVGFTQTSVL HQYFLNVFNT GMYI KSALT ALIYQKSLVL SNEASGLSST GDIVNLMSVD VQKLQDLTQW LNLIWSGPFQ IIICLYSLYK LLGNSMWVGV IILVI MMPL NSFLMRIQKK LQKSQMKYKD ERTRVISEIL NNIKSLKLYA WEKPYREKLE EVRNNKELKN LTKLGCYMAV TSFQFN IVP FLVSCCTFAV FVYTEDRALT TDLVFPALTL FNLLSFPLMI IPMVLNSFIE ASVSIGRLFT FFTNEELQPD SVQRLPK VK NIGDVAINIG DDATFLWQRK PEYKVALKNI NFQAKKGNLT CIVGKVGSGK TALLSCMLGD LFRVKGFATV HGSVAYVS Q VPWIMNGTVK ENILFGHRYD AEFYEKTIKA CALTIDLAIL MDGDKTLVGE KGISLSGGQK ARLSLARAVY ARADTYLLD DPLAAVDEHV ARHLIEHVLG PNGLLHTKTK VLATNKVSAL SIADSIALLD NGEITQQGTY DEITKDADSP LWKLLNNYGK KNNGKSNEF GDSSESSVRE SSIPVEGELE QLQKLNDLDF GNSDAISLRR A(SEP)DA(TPO)LG(SEP)ID FGDDENIAK REHREQGKVK WNIYLEYAKA CNPKSVCVFI LFIVISMFLS VMGNVWLKHW SEVNSRYGSN PNAARYLAIY FALGIGSALA TLIQTIVLW VFCTIHASKY LHNLMTNSVL RAPMTFFETT PIGRILNRFS NDIYKVDALL GRTFSQFFVN AVKVTFTITV I CATTWQFI FIIIPLSVFY IYYQQYYLRT SRELRRLDSI TRSPIYSHFQ ETLGGLATVR GYSQQKRFSH INQCRIDNNM SA FYPSINA NRWLAYRLEL IGSIIILGAA TLSVFRLKQG TLTAGMVGLS LSYALQITQT LNWIVRMTVE VETNIVSVER IKE YADLKS EAPLIVEGHR PPKEWPSQGD IKFNNYSTRY RPELDLVLKH INIHIKPNEK VGIVGRTGAG KSSLTLALFR MIEA SEGNI VIDNIAINEI GLYDLRHKLS IIPQDSQVFE GTVRENIDPI NQYTDEAIWR ALELSHLKEH VLSMSNDGLD AQLTE GGGN LSVGQRQLLC LARAMLVPSK ILVLDQATAA VDVETDKVVQ ETIRTAFKDR TILTIAHRLN TIMDSDRIIV LDNGKV AEF DSPGQLLSDN KSLFYSLCME AGLVNENGLV PRGSSAHHHH HHHHHHGA

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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

Concentration5.94 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
300.0 mMNaClSodium chloridesodium chloride
50.0 mMTristris(hydroxymethyl)aminomethane

Details: Solution were made fresh in cold distilled water and final pH was adjusted to 7.0 with HCl of cold buffer. The digitonin detergent was added to final .06 % in buffer after pH adjustment.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 283.15 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.9 µm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8499 / Average exposure time: 2.9 sec. / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2159582 / Details: Relion autopick
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER / Details: Ab-initio Model generated in Relion.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM (ver. 1.0.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0) / Number images used: 114963
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6jb1


Chain - Chain ID: B
Output model

PDB-7m69:
E1435Q Ycf1 mutant in inward-facing wide conformation

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