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- EMDB-18352: S. cerevisia Niemann-Pick type C protein NCR1 in LMNG at pH 5.5 -

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Basic information

Entry
Database: EMDB / ID: EMD-18352
TitleS. cerevisia Niemann-Pick type C protein NCR1 in LMNG at pH 5.5
Map data
Sample
  • Complex: NCR1 pH 5.5 in LMNG
    • Protein or peptide: NPC intracellular sterol transporter 1-related protein 1
  • Ligand: ERGOSTEROL
  • Ligand: PHOSPHATIDYLETHANOLAMINE
Keywordssterol transport / vacuole / lysosome / LIPID TRANSPORT / membrane protein
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
NPC intracellular sterol transporter 1-related protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsFrain KM / Nel L / Dedic E / Olesen E / Stokes D / Panyella Pedersen B
Funding support United States, Denmark, 3 items
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, 2023-
Header (metadata) releaseOct 18, 2023-
Map releaseOct 18, 2023-
UpdateApr 17, 2024-
Current statusApr 17, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18352.png

Downloads & links

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Map

FileDownload / File: emd_18352.map.gz / Format: CCP4 / Size: 59.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.302 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.0074909 - 1.7634767
Average (Standard dev.)-0.0009947063 (±0.030364761)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions250250250
Spacing250250250
CellA=B=C: 325.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18352_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18352_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18352_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : NCR1 pH 5.5 in LMNG

EntireName: NCR1 pH 5.5 in LMNG
Components
  • Complex: NCR1 pH 5.5 in LMNG
    • Protein or peptide: NPC intracellular sterol transporter 1-related protein 1
  • Ligand: ERGOSTEROL
  • Ligand: PHOSPHATIDYLETHANOLAMINE

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Supramolecule #1: NCR1 pH 5.5 in LMNG

SupramoleculeName: NCR1 pH 5.5 in LMNG / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 132.6 KDa

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Macromolecule #1: NPC intracellular sterol transporter 1-related protein 1

MacromoleculeName: NPC intracellular sterol transporter 1-related protein 1
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 132.755094 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae BY4741 (yeast)
SequenceString: MNVLWIIALV GQLMRLVQGT ATCAMYGNCG KKSVFGNELP CPVPRSFEPP VLSDETSKLL VEVCGEEWKE VRYACCTKDQ VVALRDNLQ KAQPLISSCP ACLKNFNNLF CHFTCAADQG RFVNITKVEK SKEDKDIVAE LDVFMNSSWA SEFYDSCKNI K FSATNGYA ...String:
MNVLWIIALV GQLMRLVQGT ATCAMYGNCG KKSVFGNELP CPVPRSFEPP VLSDETSKLL VEVCGEEWKE VRYACCTKDQ VVALRDNLQ KAQPLISSCP ACLKNFNNLF CHFTCAADQG RFVNITKVEK SKEDKDIVAE LDVFMNSSWA SEFYDSCKNI K FSATNGYA MDLIGGGAKN YSQFLKFLGD AKPMLGGSPF QINYKYDLAN EEKEWQEFND EVYACDDAQY KCACSDCQES CP HLKPLKD GVCKVGPLPC FSLSVLIFYT ICALFAFMWY YLCKRKKNGA MIVDDDIVPE SGSLDESETN VFESFNNETN FFN GKLANL FTKVGQFSVE NPYKILITTV FSIFVFSFII FQYATLETDP INLWVSKNSE KFKEKEYFDD NFGPFYRTEQ IFVV NETGP VLSYETLHWW FDVENFITEE LQSSENIGYQ DLCFRPTEDS TCVIESFTQY FQGALPNKDS WKRELQECGK FPVNC LPTF QQPLKTNLLF SDDDILNAHA FVVTLLLTNH TQSANRWEER LEEYLLDLKV PEGLRISFNT EISLEKELNN NNDIST VAI SYLMMFLYAT WALRRKDGKT RLLLGISGLL IVLASIVCAA GFLTLFGLKS TLIIAEVIPF LILAIGIDNI FLITHEY DR NCEQKPEYSI DQKIISAIGR MSPSILMSLL CQTGCFLIAA FVTMPAVHNF AIYSTVSVIF NGVLQLTAYV SILSLYEK R SNYKQITGNE ETKESFLKTF YFKMLTQKRL IIIIFSAWFF TSLVFLPEIQ FGLDQTLAVP QDSYLVDYFK DVYSFLNVG PPVYMVVKNL DLTKRQNQQK ICGKFTTCER DSLANVLEQE RHRSTITEPL ANWLDDYFMF LNPQNDQCCR LKKGTDEVCP PSFPSRRCE TCFQQGSWNY NMSGFPEGKD FMEYLSIWIN APSDPCPLGG RAPYSTALVY NETSVSASVF RTAHHPLRSQ K DFIQAYSD GVRISSSFPE LDMFAYSPFY IFFVQYQTLG PLTLKLIGSA IILIFFISSV FLQNIRSSFL LALVVTMIIV DI GALMALL GISLNAVSLV NLIICVGLGV EFCVHIVRSF TVVPSETKKD ANSRVLYSLN TIGESVIKGI TLTKFIGVCV LAF AQSKIF DVFYFRMWFT LIIVAALHAL LFLPALLSLF GGESYRDDSI EAED

UniProtKB: NPC intracellular sterol transporter 1-related protein 1

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Macromolecule #3: ERGOSTEROL

MacromoleculeName: ERGOSTEROL / type: ligand / ID: 3 / Number of copies: 2 / Formula: ERG
Molecular weightTheoretical: 396.648 Da
Chemical component information

ChemComp-ERG:
ERGOSTEROL

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Macromolecule #4: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 4 / Number of copies: 1 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration10 mg/mL
BufferpH: 5.5
Component:
ConcentrationFormulaName
200.0 mMNaClSodium Chloride
20.0 mMC6H13NO4S2-(N-morpholino)ethanesulfonic acid
0.005 %C47H88O22LMNG
GridModel: C-flat-1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: NITROGEN / Chamber humidity: 99 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5790 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 8541 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 853702
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 53670
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.31)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Avg.num./class: 100000 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6r4l


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 77.1
Output model

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

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