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- PDB-7mpe: Cryo-EM structure of the yeast cadmium factor 1 protein (Ycf1p) -

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Basic information

Entry
Database: PDB / ID: 7mpe
TitleCryo-EM structure of the yeast cadmium factor 1 protein (Ycf1p)
ComponentsMetal resistance protein YCF1
KeywordsTRANSPORT PROTEIN / ATP binding cassette (ABC) transporter / transmembrane domain 0 (TMD0) / regulatory (R) region
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: 3.2 Å
AuthorsBickers, S.C. / Benlekbir, S. / Rubinstein, J.L. / Kanelis, V.
Funding support Canada, 3items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 2, 2021Provider: repository / Type: Initial release
Revision 1.1May 29, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Metal resistance protein YCF1


Theoretical massNumber of molelcules
Total (without water)174,0241
Polymers174,0241
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
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: 174024.141 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / Variant: ySCB / Strain: BJ2168
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: Yeast cadmium factor 1 protein (Ycf1p) / Type: ORGANELLE OR CELLULAR COMPONENT
Details: Endogenously expressed Ycf1p that also contains a C-terminal 3x FLAG tag.
Entity ID: all / Source: NATURAL
Molecular weightValue: 0.171136 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ2168 / Cellular location: Vacoular Membrane
Buffer solutionpH: 8 / Details: Buffer was at 4 degrees Celsius.
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtris (hydroxymethyl) aminomethane hydrochlorideTris-HCl1
2150 mMsodium chlorideNaCl1
30.006 % (w/v)glyco-diosgeninGDN1
SpecimenConc.: 5.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 277 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
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9.6 sec. / Electron dose: 45 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARCLive and v.2particle selectionInitial particles were picked using the Live interface to generate a template, and the template was then used for particle selection.
2cryoSPARCLiveimage acquisitionReal time alignement, CTF fit and 2D classification.
4cryoSPARCv.2CTF correction
10cryoSPARCv.2initial Euler assignment
11cryoSPARCv.2final Euler assignment
12cryoSPARCv.2classification
13cryoSPARCv.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2789383
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124864 / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00511450
ELECTRON MICROSCOPYf_angle_d0.67315539
ELECTRON MICROSCOPYf_dihedral_angle_d9.6971536
ELECTRON MICROSCOPYf_chiral_restr0.0441820
ELECTRON MICROSCOPYf_plane_restr0.0041944

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