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- PDB-8sg4: E1435Q Ycf1 mutant in dephosphorylated state -

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Basic information

Entry
Database: PDB / ID: 8sg4
TitleE1435Q Ycf1 mutant in dephosphorylated state
ComponentsMetal resistance protein YCF1
KeywordsMEMBRANE PROTEIN / ABC transporter
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 / Atorvastatin ADME / Paracetamol 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 / Atorvastatin ADME / Paracetamol ADME / P-type cadmium transporter activity / bilirubin transmembrane transporter activity / bilirubin transport / vacuole fusion, non-autophagic / ABC-family proteins mediated transport / 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 / membrane raft / 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. ...: / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.11 Å
AuthorsKhandelwal, N.K. / Tomasiak, T.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)NIH R00 GM11424 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)NIH R01 AI156270 United States
CitationJournal: Nat Commun / Year: 2024
Title: Structural basis for autoinhibition by the dephosphorylated regulatory domain of Ycf1.
Authors: Nitesh Kumar Khandelwal / Thomas M Tomasiak /
Abstract: Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding ...Yeast Cadmium Factor 1 (Ycf1) sequesters glutathione and glutathione-heavy metal conjugates into yeast vacuoles as a cellular detoxification mechanism. Ycf1 belongs to the C subfamily of ATP Binding Cassette (ABC) transporters characterized by long flexible linkers, notably the regulatory domain (R-domain). R-domain phosphorylation is necessary for activity, whereas dephosphorylation induces autoinhibition through an undefined mechanism. Because of its transient and dynamic nature, no structure of the dephosphorylated Ycf1 exists, limiting understanding of this R-domain regulation. Here, we capture the dephosphorylated Ycf1 using cryo-EM and show that the unphosphorylated R-domain indeed forms an ordered structure with an unexpected hairpin topology bound within the Ycf1 substrate cavity. This architecture and binding mode resemble that of a viral peptide inhibitor of an ABC transporter and the secreted bacterial WXG peptide toxins. We further reveal the subset of phosphorylation sites within the hairpin turn that drive the reorganization of the R-domain conformation, suggesting a mechanism for Ycf1 activation by phosphorylation-dependent release of R-domain mediated autoinhibition.
History
DepositionApr 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Metal resistance protein YCF1


Theoretical massNumber of molelcules
Total (without water)176,2101
Polymers176,2101
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Metal resistance protein YCF1 / ABC-type Cd(2+) transporter / ABC-type glutathione-S-conjugate transporter / Yeast cadmium factor 1


Mass: 176209.594 Da / Num. of mol.: 1 / Mutation: E1435Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Strain: Saccharomyces cerevisiae / Gene: YCF1, YDR135C, YD9302.11C / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P39109, ABC-type Cd2+ transporter, ABC-type glutathione-S-conjugate transporter

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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: Ycf1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.17666831 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: DSY-5
Buffer solutionpH: 7
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.
Buffer component
IDConc.NameFormulaBuffer-ID
1300 mMsodium chlorideNaCl1
250 mMtris(hydroxymethyl)aminomethaneTris1
SpecimenConc.: 10.56 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 81000 X / Nominal defocus max: 1900 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 3 sec. / Electron dose: 52 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5904

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisitionSerialEM v4.0.0beta
4CTFFIND4.1CTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
10RELION4initial Euler assignment
11cryoSPARC4.2.1final Euler assignment
13cryoSPARC4.2.13D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5424196 / Details: Relion autopick
3D reconstructionResolution: 3.11 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 73611 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingAccession code: AF-P39109-F1 / Source name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 118.37 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.006511920
ELECTRON MICROSCOPYf_angle_d0.980716158
ELECTRON MICROSCOPYf_chiral_restr0.05951872
ELECTRON MICROSCOPYf_plane_restr0.00992020
ELECTRON MICROSCOPYf_dihedral_angle_d12.52334346

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