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- PDB-9ayc: Tetra-phosphorylated, E1435Q Ycf1 mutant in inward-facing wide co... -

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Basic information

Entry
Database: PDB / ID: 9ayc
TitleTetra-phosphorylated, E1435Q Ycf1 mutant in inward-facing wide conformation
ComponentsMetal resistance protein YCF1
KeywordsMEMBRANE PROTEIN / ABC-transporter / ycf1 / r-domain / phosphorylation / heavy metal
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 / 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 (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsCarvalho, R.S.A. / Rasel, M.S.I. / Khandelwal, N.K. / Tomasiak, T.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)NIH R01 AI156270 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)NIH S10 OD011981 United States
CitationJournal: To Be Published
Title: Cryo-EM structure of the tetra-phosphorylated R-domain in Ycf1 reveals key interactions for transport regulation
Authors: Carvalho, R.S.A. / Rasel, M.S.I. / Khandelwal, N.K. / Tomasiak, T.M.
History
DepositionMar 7, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _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)176,2631
Polymers176,2631
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: 176263.234 Da / Num. of mol.: 1 / Mutation: E1435Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
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
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: Phosphorylated, E1435Q Ycf1 mutant in inward-facing wide conformation
Type: ORGANELLE OR CELLULAR COMPONENT / Details: c-AMP PKA treated E1435Q Ycf1 mutant / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 176462 kDa/nm / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisTRIS-Hcl1
2300 mMNaClNaCl1
30.02 %GDNGDN1
SpecimenConc.: 5.94 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
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 K
Details: 5 microliters of sample of concentrated E1435Q (5.94 mg/mL) PKA-treated Ycf1 sample was applied to QF-1.2/1,3 Au grid. Grids were place inside of a Leica EM GP2 equilibrated at 10 degress ...Details: 5 microliters of sample of concentrated E1435Q (5.94 mg/mL) PKA-treated Ycf1 sample was applied to QF-1.2/1,3 Au grid. Grids were place inside of a Leica EM GP2 equilibrated at 10 degress Celcius amd 80% humidity. Following 10 seconds incubation, the side of the grid to which the sample was applied was blotted on a Whatman 1 paper (3.5 seconds) then immediately plunge frrozen in liquid ethan equilibrated at -185 degree Celcius.

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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 / Nominal defocus max: 2100 nm / Nominal defocus min: 900 nm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 0.9 sec. / Electron dose: 54 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8587
Details: Movies were collected at 22,500X magnification with automated super-resolution mode and defocus range of -0.9 to -2.1 micrometer. Moview frames cotained 60 frames with a per frame expousre ...Details: Movies were collected at 22,500X magnification with automated super-resolution mode and defocus range of -0.9 to -2.1 micrometer. Moview frames cotained 60 frames with a per frame expousre of 0.9 electrons per angstrom squared (54 electron/angstrom square total dose)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
2cryoSPARCimage acquisition
4cryoSPARC4.2.1CTF correction
7ISOLDE1.3model fitting
9PHENIX1.20.1model refinement
13cryoSPARC4.2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4840688
Details: Manual and auto particle picking and extraction from 8380 curated micropgrahs
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68169 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Atomic model buildingAccession code: p39109 / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412022
ELECTRON MICROSCOPYf_angle_d0.58816299
ELECTRON MICROSCOPYf_dihedral_angle_d11.324378
ELECTRON MICROSCOPYf_chiral_restr0.0391883
ELECTRON MICROSCOPYf_plane_restr0.0042036

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