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

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Basic information

Entry
Database: PDB / ID: 7m69
TitleE1435Q Ycf1 mutant in inward-facing wide conformation
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 / ABC-family proteins mediated transport / vacuole fusion, non-autophagic / ABC-type glutathione S-conjugate transporter activity / ABC-type glutathione-S-conjugate transporter / fungal-type vacuole / 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 TMD0 domain / : / 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 TMD0 domain / : / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.42 Å
AuthorsKhandelwal, N.K. / Millan, C.R. / Thaker, T.M. / Tomasiak, T.M.
Funding support United States, 1items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 6, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 19, 2022Group: Database references / Category: citation / citation_author
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
Revision 1.2Oct 16, 2024Group: Data collection / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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)176,3181
Polymers176,3181
Non-polymers00
Water00
1


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

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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: 176318.328 Da / Num. of mol.: 1 / Mutation: E1435Q
Source method: isolated from a genetically manipulated source
Details: Phosphorylated residues (S908, T911 and S914) are present in the structure file.
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: YCF1, YDR135C, YD9302.11C / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): DSY-5
References: UniProt: P39109, ABC-type Cd2+ transporter, ABC-type glutathione-S-conjugate transporter
Has ligand of interestY
Has protein modificationY

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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.: 5.94 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: 22500 X / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 2.9 sec. / Electron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8499

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.1_4122refinement
PHENIX1.19.1_4122refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisitionscript LD
4CTFFIND4.1CTF correction
7ISOLDEmodel fitting
8Cootmodel fitting
10RELION3.1.1initial Euler assignment
11cisTEM1.0.0final Euler assignment
13cisTEM1.0.03D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2159582 / Details: Relion autopick
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.42 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 114963 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6JB1

6jb1


Pdb chain-ID: B / Accession code: 6JB1 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 146.4 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005411100
ELECTRON MICROSCOPYf_angle_d0.855315064
ELECTRON MICROSCOPYf_chiral_restr0.04411774
ELECTRON MICROSCOPYf_plane_restr0.00511876
ELECTRON MICROSCOPYf_dihedral_angle_d13.32573981

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