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

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Basic information

Entry
Database: PDB / ID: 3jd8
Titlecryo-EM structure of the full-length human NPC1 at 4.4 angstrom
ComponentsNiemann-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 / 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 / adult walking behavior / cholesterol efflux / 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 / gene expression / late endosome membrane / lysosome / response to xenobiotic stimulus / symbiont entry into host cell / membrane raft / lysosomal 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 / Sterol-sensing domain of SREBP cleavage-activation / Protein patched/dispatched / Patched family / Sterol-sensing domain (SSD) profile. / Sterol-sensing domain
Similarity search - Domain/homology
CHOLESTEROL / NPC intracellular cholesterol transporter 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.43 Å
AuthorsGong, X. / Qian, H.W. / Zhou, X.H. / Wu, J.P. / 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, 2016Deposition site: RCSB / Processing site: PDBJ
Revision 1.0Jun 1, 2016Provider: repository / Type: Initial release
Revision 1.1Jun 8, 2016Group: Other
Revision 1.2Jun 13, 2018Group: Advisory / Data collection ...Advisory / Data collection / Database references / Source and taxonomy
Category: database_2 / em_image_scans ...database_2 / em_image_scans / em_software / entity_src_gen / pdbx_unobs_or_zero_occ_atoms
Item: _em_software.name / _entity_src_gen.host_org_common_name ..._em_software.name / _entity_src_gen.host_org_common_name / _entity_src_gen.pdbx_host_org_cell_line / _entity_src_gen.pdbx_host_org_strain / _pdbx_unobs_or_zero_occ_atoms.label_asym_id
Revision 1.3Oct 9, 2019Group: Data collection / Derived calculations / Other / Category: cell / struct_conn
Item: _cell.length_a / _cell.length_b ..._cell.length_a / _cell.length_b / _cell.length_c / _struct_conn.pdbx_leaving_atom_flag
Revision 1.4Dec 18, 2019Group: Data collection / Category: em_software
Item: _em_software.fitting_id / _em_software.image_processing_id
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / 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 / _chem_comp.name / _chem_comp.type / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_unobs_or_zero_occ_atoms.auth_asym_id / _pdbx_unobs_or_zero_occ_atoms.auth_seq_id / _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_comp_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Niemann-Pick C1 protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,70118
Polymers142,2731
Non-polymers5,42817
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

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Protein / Non-polymers , 2 types, 2 molecules A

#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 / Plasmid: pCAG / Cell line (production host): HEK-293F / Production host: Homo sapiens (human) / References: UniProt: O15118
#6: Chemical ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O

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Sugars , 4 types, 16 molecules

#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.: 3
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
#3: 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-ROHGlycam 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
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE
#4: 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-ROHGlycam 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
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#5: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 11
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

ComponentName: Niemann-Pick C1 protein / Type: COMPLEX / Synonym: NPC1
Buffer solutionName: 25mM Tris pH 8.0, 150mM NaCl and 0.1% digitonin / pH: 8 / Details: 25mM Tris pH 8.0, 150mM NaCl and 0.1% digitonin
SpecimenConc.: 15 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil R1.2/1.3 copper grid, 200 mesh
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Jan 10, 2016
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: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderTemperature: 100 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1UCSF Chimeramodel fitting
2RELION1.43D reconstruction
CTF correctionDetails: Each micrograph
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: RELIONList of Walmart brands / Resolution: 4.43 Å / Num. of particles: 102731 / Nominal pixel size: 1.30654 Å / Actual pixel size: 1.30654 Å
Details: (Single particle details: Iamges were processed using RELION1.4.) (Single particle--Applied symmetry: C1)
Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALREFINEMENT PROTOCOL--rigid body
2RIGID BODY FITREALREFINEMENT PROTOCOL--rigid body
Atomic model building
IDPDB-ID 3D fitting-ID
15F1B

5f1b

1
23GKI

3gki

2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms7694 0 355 0 8049

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