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- PDB-8qec: S. cerevisia Niemann-Pick type C protein NCR1 in GDN at pH 5.5 -

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Basic information

Entry
Database: PDB / ID: 8qec
TitleS. cerevisia Niemann-Pick type C protein NCR1 in GDN at pH 5.5
ComponentsNPC intracellular sterol transporter 1-related protein 1
KeywordsMEMBRANE PROTEIN / sterol transport / vacuole / lysosome / LIPID TRANSPORT
Function / homology
Function and homology information


Intestinal lipid absorption / LDL clearance / sterol transport / sterol binding / sphingolipid metabolic process / fungal-type vacuole membrane / cholesterol binding / cholesterol homeostasis / endoplasmic reticulum / membrane / plasma membrane
Similarity search - Function
Niemann-Pick C1, N-terminal / Niemann-Pick C1 N terminus / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
ERGOSTEROL / Chem-Q7G / CHOLESTEROL HEMISUCCINATE / NPC intracellular sterol transporter 1-related protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsFrain, K.M. / Dedic, E. / Nel, L. / Olesen, E. / Stokes, D. / Panyella Pedersen, B.
Funding support United States, Denmark, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM144109 United States
Danish Council for Independent Research0135-00032B Denmark
The Carlsberg FoundationCF19-0127 Denmark
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.
Authors: Kelly M Frain / Emil Dedic / Lynette Nel / Anastasiia Bohush / Esben Olesen / Katja Thaysen / Daniel Wüstner / David L Stokes / Bjørn Panyella Pedersen /
Abstract: The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. NCR1 is localized to the vacuolar membrane, where it is suggested to carry ...The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.
History
DepositionAug 31, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 18, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2024Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: NPC intracellular sterol transporter 1-related protein 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)136,9889
Polymers132,7551
Non-polymers4,2338
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 NPC intracellular sterol transporter 1-related protein 1


Mass: 132755.094 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: NCR1 / Production host: Saccharomyces cerevisiae BY4741 (yeast) / References: UniProt: Q12200
#2: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#3: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-ERG / ERGOSTEROL


Mass: 396.648 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H44O / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-Q7G / 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl}-4-{[(3beta,9beta,14beta,17beta,25R)-spirost-5-en-3-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranoside


Mass: 1165.315 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C56H92O25
Has ligand of interestY

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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: NCR1 in GDN at pH 5.5 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1326 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BY4741
Buffer solutionpH: 5.5
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMSodium chlorideNaCl1
220 mMMESC6H13NO4S1
30.01 %GDNC56H92O251
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 99 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2600 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6036
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2EPUimage acquisition
4cryoSPARC3.3.1CTF correction
7UCSF ChimeraXmodel fitting
9PHENIXmodel refinement
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1888836
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 496228 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 179.2 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6R4L

6r4l


Pdb chain-ID: A / Accession code: 6R4L / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0029342
ELECTRON MICROSCOPYf_angle_d0.53512715
ELECTRON MICROSCOPYf_dihedral_angle_d8.6651332
ELECTRON MICROSCOPYf_chiral_restr0.0811478
ELECTRON MICROSCOPYf_plane_restr0.0031572

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