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- PDB-6r4l: Crystal structure of S. cerevisia Niemann-Pick type C protein NCR1 -

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Basic information

Entry
Database: PDB / ID: 6r4l
TitleCrystal structure of S. cerevisia Niemann-Pick type C protein NCR1
ComponentsNPC intracellular cholesterol transporter 1-related protein 1
KeywordsLIPID TRANSPORT / Vacuole / Ergosterol / 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
ERGOSTEROL / NPC intracellular sterol transporter 1-related protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 3.5 Å
AuthorsWinkler, M.B.L. / Kidmose, R.T. / Pedersen, B.P.
Funding support Denmark, 4items
OrganizationGrant numberCountry
European Research Council637372 Denmark
Danish Council for Independent ResearchDFF-4002-0052 Denmark
Other privateCarlsberg Foundation CF17-0180 Denmark
Other privateAIAS fellowship Denmark
CitationJournal: Cell / Year: 2019
Title: Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins.
Authors: Mikael B L Winkler / Rune T Kidmose / Maria Szomek / Katja Thaysen / Shaun Rawson / Stephen P Muench / Daniel Wüstner / Bjørn Panyella Pedersen /
Abstract: Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, ...Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, and biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system (NCR1 and NPC2), we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration that clarifies the role of NPC proteins in this essential eukaryotic pathway and that rationalizes mutations in patients with Niemann-Pick disease type C.
History
DepositionMar 22, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 25, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 2, 2019Group: Data collection / 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.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Oct 16, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...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 / 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.occupancy / _atom_site.type_symbol / _chem_comp.name / _chem_comp.type / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.src_method / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Jan 24, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _chem_comp.pdbx_synonyms / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)137,5046
Polymers135,4921
Non-polymers2,0125
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2510 Å2
ΔGint24 kcal/mol
Surface area57660 Å2
MethodPISA
Unit cell
Length a, b, c (Å)148.670, 90.060, 161.680
Angle α, β, γ (deg.)90.000, 110.310, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Protein NPC intracellular cholesterol transporter 1-related protein 1 / Niemann-Pick type C-related protein 1


Mass: 135491.844 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: NCR1, YPL006W / Plasmid: p423_GAL1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): DSY-5 / References: UniProt: Q12200
#2: 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.: 2
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
#3: Chemical ChemComp-ERG / ERGOSTEROL


Mass: 396.648 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C28H44O
#4: 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


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.75 Å3/Da / Density % sol: 67.16 %
Crystal growTemperature: 291 K / Method: lipidic cubic phase / pH: 6.1
Details: MES, Ammonium Chloride, Manganese Chloride, PEP 426

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9795 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 29, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9795 Å / Relative weight: 1
ReflectionResolution: 3.3→49.124 Å / Num. obs: 28581 / % possible obs: 93.9 % / Redundancy: 1.802 % / Biso Wilson estimate: 134.51 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.066 / Rrim(I) all: 0.089 / Χ2: 1.043 / Net I/σ(I): 7.05
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
3.3-3.41.8142.1470.4424490.1722.89295.7
3.4-3.51.7522.0780.5122010.2542.81295
3.5-3.61.8221.3510.7720000.4211.82296.5
3.6-3.71.8180.9311.1217630.5531.25597.1
3.7-3.81.8180.6821.5115850.6760.92296.2
3.8-3.91.7240.4631.8914270.7990.62696.4
3.9-41.7530.3842.412650.8960.51796.1
4-4.51.8210.2134.3147880.9640.28694
4.5-51.8180.1137.6530660.9840.15294.3
5-61.7820.0968.6134420.9880.1393.3
6-71.8320.06312.2117290.9930.08591.9
7-81.7740.03520.19620.9970.04889.3
8-101.8280.01932.779360.9990.02687.7
10-151.8440.01442.776940.9990.01986.5
15-201.8430.01246.611660.9990.01784.3
20-49.1241.7870.01150.3910810.01668.8

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation7.26 Å49.18 Å
Translation7.26 Å49.18 Å

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
XDSdata reduction
XSCALEdata scaling
PHASER2.8.1phasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5U73, 3GKH

5u73

3gkh


Resolution: 3.5→49.124 Å / SU ML: 0.55 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 38.38
RfactorNum. reflection% reflection
Rfree0.3012 1209 5.07 %
Rwork0.2656 --
obs0.2675 23868 93.56 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 396.41 Å2 / Biso mean: 157.7351 Å2 / Biso min: 57.11 Å2
Refinement stepCycle: final / Resolution: 3.5→49.124 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8759 0 135 0 8894
Biso mean--217.82 --
Num. residues----1096
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.019137
X-RAY DIFFRACTIONf_angle_d1.10912431
X-RAY DIFFRACTIONf_chiral_restr0.0611423
X-RAY DIFFRACTIONf_plane_restr0.0081556
X-RAY DIFFRACTIONf_dihedral_angle_d12.2045400
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 9

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.5001-3.64020.40081390.36942562270196
3.6402-3.80580.37611360.34192566270297
3.8058-4.00630.34421360.32832564270096
4.0063-4.25720.34321370.30332541267894
4.2572-4.58570.33821330.26672501263494
4.5857-5.04680.33461460.26822487263393
5.0468-5.77610.37441470.29232529267694
5.7761-7.27340.38481080.31232497260592
7.2734-49.12840.19991270.19842412253987
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
1-0.05150.58131.73683.79442.91743.787-0.0904-0.1852-0.06430.5146-0.07770.45790.4461-0.41870.08460.95370.02720.03670.97260.10321.1655-32.86925.555137.8827
23.12440.37891.02283.3111.072.8197-0.18790.14910.0337-0.39550.23090.2281-0.271-0.32410.01080.9781-0.0577-0.00370.52330.10730.622-31.751421.6894-0.792
31.85370.20131.84962.13080.36276.3928-0.33610.1171-0.0056-0.06190.2526-0.0776-0.69970.78120.10610.5766-0.08180.03710.54770.05710.7733-15.232329.801128.7858
43.78120.1398-1.27910.97270.2950.5999-0.3026-1.62250.72940.48880.30840.23680.87890.12310.24221.8078-0.3327-0.15552.229-0.32891.46030.303262.984383.4127
52.5148-1.4533-0.69181.14110.7455.16140.4382-0.14050.4671-0.3376-0.7440.7168-0.5286-0.40690.49411.9221-0.2966-0.111.6429-0.27041.682-7.151359.313870.2449
60.25980.42981.39022.08662.95854.16960.42-0.08520.6631-0.47570.5144-0.8538-0.2545-0.2039-0.61871.3515-0.30580.08552.0516-0.13722.3376-3.249759.988556.3662
72.7013-0.8888-0.86986.2165-3.10272.2156-1.35752.77440.6314-3.61192.29822.0161-1.00772.739-0.96883.3629-1.3399-0.46592.6026-0.12562.0279-5.661251.8112-3.2494
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1chain 'A' and (resid 316 through 546)A316 - 546
2X-RAY DIFFRACTION2chain 'A' and (resid 547 through 796)A547 - 796
3X-RAY DIFFRACTION3chain 'A' and (resid 797 through 1159 )A797 - 1159
4X-RAY DIFFRACTION4chain 'A' and (resid 21 through 77 )A21 - 77
5X-RAY DIFFRACTION5chain 'A' and (resid 78 through 178 )A78 - 178
6X-RAY DIFFRACTION6chain 'A' and (resid 179 through 256 )A179 - 256
7X-RAY DIFFRACTION7chain 'A' and (resid 257 through 279 )A257 - 279

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