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- PDB-5jnx: The 6.6 A cryo-EM structure of the full-length human NPC1 in comp... -

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Entry
Database: PDB / ID: 5jnx
TitleThe 6.6 A cryo-EM structure of the full-length human NPC1 in complex with the cleaved glycoprotein of Ebola virus
Components
  • (Envelope glycoprotein) x 2
  • Niemann-Pick C1 protein
KeywordsMEMBRANE PROTEIN / protein complex
Function / homology
Function and homology information


cyclodextrin metabolic process / cholesterol storage / membrane raft organization / : / intracellular cholesterol transport / intracellular lipid transport / intestinal cholesterol absorption / LDL clearance / programmed cell death / negative regulation of epithelial cell apoptotic process ...cyclodextrin metabolic process / cholesterol storage / membrane raft organization / : / intracellular cholesterol transport / intracellular lipid transport / intestinal cholesterol absorption / LDL clearance / programmed cell death / negative regulation of epithelial cell apoptotic process / cholesterol transport / bile acid metabolic process / establishment of protein localization to membrane / cholesterol efflux / adult walking behavior / lysosomal transport / cholesterol binding / negative regulation of macroautophagy / cellular response to steroid hormone stimulus / protein glycosylation / cellular response to low-density lipoprotein particle stimulus / response to cadmium ion / negative regulation of TORC1 signaling / cholesterol metabolic process / neurogenesis / cholesterol homeostasis / liver development / macroautophagy / autophagy / endocytosis / transmembrane signaling receptor activity / virus receptor activity / signaling receptor activity / nuclear envelope / late endosome membrane / gene expression / clathrin-dependent endocytosis of virus by host cell / suppression by virus of host tetherin activity / entry receptor-mediated virion attachment to host cell / lysosome / response to xenobiotic stimulus / symbiont entry into host cell / membrane raft / lysosomal membrane / fusion of virus membrane with host endosome membrane / viral envelope / lipid binding / symbiont-mediated suppression of host type I interferon-mediated signaling pathway / host cell plasma membrane / virion membrane / perinuclear region of cytoplasm / Golgi apparatus / endoplasmic reticulum / extracellular exosome / extracellular region / membrane / plasma membrane
Similarity search - Function
NPC1-like / Niemann-Pick C1, N-terminal / Niemann-Pick C1 N terminus / Filoviruses glycoprotein, extracellular domain / Filoviruses glycoprotein / Filovirus glycoprotein / Sterol-sensing domain of SREBP cleavage-activation / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
NPC intracellular cholesterol transporter 1 / Envelope glycoprotein
Similarity search - Component
Biological speciesHomo sapiens (human)
Ebola virus - Zaire
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.56 Å
AuthorsGong, X. / Qian, H.W. / Zhou, X.H. / Wu, J.P. / Wan, T. / Shi, Y. / Gao, F. / Zhou, Q. / Yan, N.
Funding support China, 3items
OrganizationGrant numberCountry
Ministry of Science and Technology of China2015CB9101012014, ZX09507003006 China
National Natural Science Foundation of Chinaproject 31321062 and 81590761 China
President Foundation of Chinese Academy of Sciences and Strategic Priority Research Program of the Chinese Academy of SciencesXDB08020100 China
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, 2016Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Jun 15, 2016Provider: repository / Type: Initial release
Revision 1.1Dec 13, 2017Group: Data processing / Database references / Category: em_software / pdbx_database_related / Item: _em_software.name
Revision 1.2Oct 23, 2019Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations
Category: pdbx_database_related / pdbx_validate_close_contact / struct_conn
Item: _pdbx_database_related.db_id
Revision 1.3Dec 4, 2019Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.energyfilter_name
Revision 1.4Dec 11, 2019Group: Data collection / Category: em_imaging_optics
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / atom_site_anisotrop ...atom_site / atom_site_anisotrop / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _atom_site_anisotrop.id / _atom_site_anisotrop.pdbx_auth_asym_id / _atom_site_anisotrop.pdbx_auth_seq_id / _atom_site_anisotrop.pdbx_label_asym_id / _chem_comp.name / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_unobs_or_zero_occ_atoms.label_asym_id / _pdbx_validate_close_contact.auth_asym_id_2 / _pdbx_validate_close_contact.auth_seq_id_2 / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Aug 5, 2020Group: Derived calculations / Source and taxonomy / Structure summary
Category: chem_comp / em_entity_assembly ...chem_comp / em_entity_assembly / em_entity_assembly_naturalsource / em_entity_assembly_recombinant / entity_src_gen / struct_conn
Item: _chem_comp.pdbx_synonyms / _entity_src_gen.gene_src_strain ..._chem_comp.pdbx_synonyms / _entity_src_gen.gene_src_strain / _entity_src_gen.pdbx_gene_src_scientific_name / _struct_conn.pdbx_leaving_atom_flag

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Niemann-Pick C1 protein
C: Envelope glycoprotein
D: Envelope glycoprotein
E: Envelope glycoprotein
F: Envelope glycoprotein
G: Envelope glycoprotein
H: Envelope glycoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)244,80225
Polymers238,5057
Non-polymers6,29718
Water0
1
A: Niemann-Pick C1 protein
C: Envelope glycoprotein
E: Envelope glycoprotein
G: Envelope glycoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,88019
Polymers193,8574
Non-polymers5,02415
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2
D: Envelope glycoprotein
F: Envelope glycoprotein
H: Envelope glycoprotein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,9226
Polymers44,6483
Non-polymers1,2733
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 7 molecules ACEGDFH

#1: Protein Niemann-Pick C1 protein


Mass: 142272.906 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: NPC1 / Cell line (production host): HEK 293F / Production host: Homo sapiens (human) / References: UniProt: O15118
#2: Protein Envelope glycoprotein / GP1 / 2 / GP


Mass: 17194.543 Da / Num. of mol.: 3 / Fragment: UNP residues 32-188 / Mutation: T42V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ebola virus - Zaire (1995) / Gene: GP / Cell line (production host): Hi-5
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P87666
#3: Protein Envelope glycoprotein / GP1 / 2 / GP


Mass: 14882.807 Da / Num. of mol.: 3 / Fragment: UNP residues 509-632
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ebola virus - Zaire (1995) / Gene: GP / Cell line (production host): Hi-5
Production host: Insect cell expression vector pTIE1 (others)
References: UniProt: P87666

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Sugars , 4 types, 18 molecules

#4: 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.: 7
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam 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
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#5: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#6: Polysaccharide alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1- ...alpha-D-mannopyranose-(1-6)-beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 748.682 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-6DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,4,3/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3/a4-b1_b4-c1_c6-d1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
#7: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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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

Component
IDNameTypeEntity IDParent-IDSource
1Niemann-Pick C1 protein in complex with GPclCOMPLEX#1-#30RECOMBINANT
2Niemann-Pick C1 proteinCOMPLEX#11RECOMBINANT
3Envelope glycoproteinCOMPLEX#2-#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Ebola virus - Zaire (1995)128951
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
22Homo sapiens (human)9606pCAG
33Insect cell expression vector pTIE1 (others)266783
Buffer solutionpH: 8
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 25 mM Tris pH 8.0, 150 mM NaCl and 0.1% digitonin
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 38270 X / Nominal defocus max: 2700 nm / Nominal defocus min: 1700 nm / Calibrated defocus min: 1700 nm / Calibrated defocus max: 2700 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 80 K
Image recordingAverage exposure time: 8 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1379
Image scansSampling size: 5 µm / Movie frames/image: 32 / Used frames/image: 0-32

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Processing

EM software
IDNameVersionCategory
1RELION1.4particle selection
2UCSFImage4image acquisition
4Gctf0.38CTF correction
10RELION1.4initial Euler assignment
11RELION1.4final Euler assignment
12RELION1.4classification
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 703336
3D reconstructionResolution: 6.56 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50223 / Symmetry type: POINT

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