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- EMDB-6640: cryo-EM map of the full-length human NPC1 at 4.4 angstrom -

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Basic information

Entry
Database: EMDB / ID: EMD-6640
Titlecryo-EM map of the full-length human NPC1 at 4.4 angstrom
Map dataReconstruction of a memebrane protein
Sample
  • Sample: NPC1
  • Protein or peptide: Niemann-Pick C1 protein
Keywordsmembrane protein
Function / homology
Function and homology information


cyclodextrin metabolic process / cholesterol storage / membrane raft organization / intracellular cholesterol transport / intracellular lipid transport / sterol transport / intestinal cholesterol absorption / LDL clearance / negative regulation of epithelial cell apoptotic process / programmed cell death ...cyclodextrin metabolic process / cholesterol storage / membrane raft organization / intracellular cholesterol transport / intracellular lipid transport / sterol transport / intestinal cholesterol absorption / LDL clearance / negative regulation of epithelial cell apoptotic process / programmed cell death / cholesterol transfer activity / cholesterol transport / bile acid metabolic process / establishment of protein localization to membrane / adult walking behavior / cholesterol efflux / lysosomal transport / cholesterol binding / cellular response to steroid hormone stimulus / negative regulation of macroautophagy / : / cellular response to low-density lipoprotein particle stimulus / response to cadmium ion / cholesterol metabolic process / negative regulation of TORC1 signaling / neurogenesis / cholesterol homeostasis / macroautophagy / liver development / autophagy / endocytosis / late endosome membrane / transmembrane signaling receptor activity / nuclear envelope / signaling receptor activity / virus receptor activity / gene expression / lysosome / membrane raft / response to xenobiotic stimulus / lysosomal membrane / symbiont entry into host cell / perinuclear region of cytoplasm / endoplasmic reticulum / Golgi apparatus / extracellular exosome / extracellular region / membrane / plasma membrane
Similarity search - Function
NPC1-like / Niemann-Pick C1, N-terminal / : / Niemann-Pick C1 N terminus / NPC1, middle luminal domain / : / Sterol-sensing domain of SREBP cleavage-activation / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
NPC intracellular cholesterol transporter 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.43 Å
AuthorsGong X / Qiang HW / Zhou XH / Wu JP / Zhou Q / Yan N
CitationJournal: Cell / Year: 2016
Title: Structural Insights into the Niemann-Pick C1 (NPC1)-Mediated Cholesterol Transfer and Ebola Infection.
Authors: Xin Gong / Hongwu Qian / Xinhui Zhou / Jianping Wu / Tao Wan / Pingping Cao / Weiyun Huang / Xin Zhao / Xudong Wang / Peiyi Wang / Yi Shi / George F Gao / Qiang Zhou / Nieng Yan /
Abstract: Niemann-Pick disease type C (NPC) is associated with mutations in NPC1 and NPC2, whose gene products are key players in the endosomal/lysosomal egress of low-density lipoprotein-derived cholesterol. ...Niemann-Pick disease type C (NPC) is associated with mutations in NPC1 and NPC2, whose gene products are key players in the endosomal/lysosomal egress of low-density lipoprotein-derived cholesterol. NPC1 is also the intracellular receptor for Ebola virus (EBOV). Here, we present a 4.4 Å structure of full-length human NPC1 and a low-resolution reconstruction of NPC1 in complex with the cleaved glycoprotein (GPcl) of EBOV, both determined by single-particle electron cryomicroscopy. NPC1 contains 13 transmembrane segments (TMs) and three distinct lumenal domains A (also designated NTD), C, and I. TMs 2-13 exhibit a typical resistance-nodulation-cell division fold, among which TMs 3-7 constitute the sterol-sensing domain conserved in several proteins involved in cholesterol metabolism and signaling. A trimeric EBOV-GPcl binds to one NPC1 monomer through the domain C. Our structural and biochemical characterizations provide an important framework for mechanistic understanding of NPC1-mediated intracellular cholesterol trafficking and Ebola virus infection.
History
DepositionMay 1, 2016-
Header (metadata) releaseJun 1, 2016-
Map releaseJun 1, 2016-
UpdateJun 8, 2016-
Current statusJun 8, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-3jd8
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6640.gif

Downloads & links

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Map

FileDownload / File: emd_6640.map.gz / Format: CCP4 / Size: 29.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a memebrane protein
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.31 Å/pix.
x 200 pix.
= 261.308 Å		1.31 Å/pix.
x 200 pix.
= 261.308 Å		1.31 Å/pix.
x 200 pix.
= 261.308 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.30654 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.03710028 - 0.08669025
Average (Standard dev.)-0.00004746 (±0.00520903)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 261.308 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.306541.306541.30654
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z261.308261.308261.308
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0370.087-0.000

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Supplemental data

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Sample components

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Entire : NPC1

EntireName: NPC1
Components
  • Sample: NPC1
  • Protein or peptide: Niemann-Pick C1 protein

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Supramolecule #1000: NPC1

SupramoleculeName: NPC1 / type: sample / ID: 1000 / Details: The sample was monodisperse / Number unique components: 1
Molecular weightExperimental: 140 KDa / Theoretical: 140 KDa

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Macromolecule #1: Niemann-Pick C1 protein

MacromoleculeName: Niemann-Pick C1 protein / type: protein_or_peptide / ID: 1 / Name.synonym: NPC1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / Organelle: lysosome / Location in cell: lysosome
Molecular weightTheoretical: 140 KDa
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK 293F / Recombinant plasmid: pCAG
SequenceUniProtKB: NPC intracellular cholesterol transporter 1

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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

Concentration15 mg/mL
BufferpH: 8 / Details: 25mM Tris pH 8.0,150mM NaCl, 0.1% digitonin
GridDetails: Quantifoil R1.2/1.3 copper grid, 200 mesh
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3-3.5 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80 K / Max: 105 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 22,500 times magnification
DateJan 10, 2016
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 4026 / Average electron dose: 50 e/Å2
Details: Every image is the average of 32 frames recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38270 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsIamges were processed using RELION1.4.
CTF correctionDetails: Each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 4.43 Å / Resolution method: OTHER / Software - Name: RELION1.4 / Number images used: 102731
Final two d classificationNumber classes: 2

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Atomic model buiding 1

Initial modelPDB ID:

5f1b


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jd8:
cryo-EM structure of the full-length human NPC1 at 4.4 angstrom

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Atomic model buiding 2

Initial modelPDB ID:

3gki


Chain - Chain ID: A
SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3jd8:
cryo-EM structure of the full-length human NPC1 at 4.4 angstrom

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