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- PDB-6r4m: Crystal structure of S. cerevisia Niemann-Pick type C protein NPC2 -

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Basic information

Entry
Database: PDB / ID: 6r4m
TitleCrystal structure of S. cerevisia Niemann-Pick type C protein NPC2
ComponentsPhosphatidylglycerol/phosphatidylinositol transfer protein
KeywordsLIPID TRANSPORT / Vacuole / Ergosterol
Function / homology
Function and homology information


fungal-type vacuole lumen / intracellular sterol transport / LDL clearance / sterol binding / sterol transport / phosphatidylcholine binding / fungal-type vacuole / phosphatidylserine binding / Neutrophil degranulation / phosphatidylinositol binding
Similarity search - Function
ML domain, phosphatidylinositol/phosphatidylglycerol transfer protein / GM2-AP, lipid-recognition domain superfamily / Sterol transport protein NPC2-like / ML domain / MD-2-related lipid-recognition domain / Domain involved in innate immunity and lipid metabolism. / Immunoglobulin E-set
Similarity search - Domain/homology
Phosphatidylglycerol/phosphatidylinositol transfer protein
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SIRAS / molecular replacement / Resolution: 2.8 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 1.3Jul 29, 2020Group: Data collection / Derived calculations ...Data collection / Derived calculations / Refinement description / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / refine / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _refine.pdbx_diffrn_id / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.4Oct 23, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp / chem_comp_atom ...chem_comp / chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
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: Phosphatidylglycerol/phosphatidylinositol transfer protein
B: Phosphatidylglycerol/phosphatidylinositol transfer protein
C: Phosphatidylglycerol/phosphatidylinositol transfer protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,1056
Polymers65,4413
Non-polymers6643
Water00
1
A: Phosphatidylglycerol/phosphatidylinositol transfer protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,0352
Polymers21,8141
Non-polymers2211
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Phosphatidylglycerol/phosphatidylinositol transfer protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,0352
Polymers21,8141
Non-polymers2211
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
C: Phosphatidylglycerol/phosphatidylinositol transfer protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,0352
Polymers21,8141
Non-polymers2211
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)205.320, 205.320, 39.080
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number169
Space group name H-MP61
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11chain A
21(chain B and resid 31 through 172)
31(chain C and resid 31 through 172)

NCS domain segments:
Dom-IDComponent-IDEns-IDSelection detailsAuth asym-IDAuth seq-ID
111chain AA31 - 175
211(chain B and resid 31 through 172)B31 - 172
311(chain C and resid 31 through 172)C31 - 172

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Components

#1: Protein Phosphatidylglycerol/phosphatidylinositol transfer protein / PG/PI-TP / NPC2 homolog


Mass: 21813.635 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Strain: ATCC 204508 / S288c / Gene: NPC2, YDL046W, D2699 / Plasmid: p423_GAL1 / Production host: Saccharomyces cerevisiae (brewer's yeast) / Strain (production host): DSY-5 / References: UniProt: Q12408
#2: 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.: 3
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
Has protein modificationY

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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: 3.63 Å3/Da / Density % sol: 66.15 %
Crystal growTemperature: 292 K / Method: vapor diffusion, sitting drop / pH: 5.1 / Details: Ammoniumcitrate dibasic, PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID23-1 / Wavelength: 0.97242 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Apr 9, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97242 Å / Relative weight: 1
ReflectionResolution: 2.8→44.453 Å / Num. obs: 23790 / % possible obs: 99.5 % / Redundancy: 3.74 % / Biso Wilson estimate: 90.35 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.081 / Rrim(I) all: 0.095 / Χ2: 1.093 / Net I/σ(I): 9.15 / Num. measured all: 88986
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsCC1/2Rrim(I) all% possible all
2.8-2.93.2331.4150.737461232123080.3721.69499.4
2.9-34.1291.0521.168596208320820.5881.212100
3-3.13.990.6961.766990175317520.7330.80499.9
3.1-3.24.0360.4462.86458160116000.8710.51599.9
3.2-3.33.9120.3453.635493140514040.9040.40199.9
3.3-3.43.6220.2744.394379121012090.9250.32299.9
3.4-3.54.0170.2385.314551113411330.9570.27599.9
3.5-3.64.1020.1857.0940499879870.9740.213100
3.6-3.73.9140.1458.3633668608600.9810.169100
3.7-3.83.9740.1359.0932158108090.9880.15699.9
3.8-3.94.0140.11810.5228667157140.9860.13699.9
3.9-43.6990.10811.3423606426380.9860.12699.4
4-53.6380.06516.414489401239830.9950.07799.3
5-63.4250.05618.976008178317540.9950.06698.4
6-83.5320.04820.495129146514520.9960.05699.1
8-103.3490.03622.2617555285240.9980.04399.2
10-153.1890.03222.6512634003960.9980.03899
15-203.0280.0322.993301091090.9970.036100
20-44.45330.02723.5122885760.9990.03189.4

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation6.8 Å44.45 Å
Translation6.8 Å44.45 Å

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Processing

Software
NameVersionClassification
PHENIXrefinement
XDS20180126data reduction
XSCALE20180808data scaling
MLPHAREphasing
PDB_EXTRACT3.24data extraction
RefinementMethod to determine structure: SIRAS / Resolution: 2.8→44.453 Å / SU ML: 0.44 / Cross valid method: THROUGHOUT / σ(F): 1.36 / Phase error: 31.03 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2618 1195 5.03 %
Rwork0.2363 22579 -
obs0.2376 23774 99.54 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 216.14 Å2 / Biso mean: 107.6192 Å2 / Biso min: 58.63 Å2
Refinement stepCycle: final / Resolution: 2.8→44.453 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3389 0 42 0 3431
Biso mean--172.27 --
Num. residues----435
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0073510
X-RAY DIFFRACTIONf_angle_d1.2494806
X-RAY DIFFRACTIONf_chiral_restr0.066564
X-RAY DIFFRACTIONf_plane_restr0.012638
X-RAY DIFFRACTIONf_dihedral_angle_d10.9752163
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDNumberRefine-IDRmsType
11A2098X-RAY DIFFRACTION12.205TORSIONAL
12B2098X-RAY DIFFRACTION12.205TORSIONAL
13C2098X-RAY DIFFRACTION12.205TORSIONAL
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 (%)
2.8-2.91210.39941280.36452434256299
2.9121-3.04460.36971320.346325002632100
3.0446-3.2050.37391320.324922624100
3.205-3.40580.28721330.268624742607100
3.4058-3.66860.30571350.25425182653100
3.6686-4.03760.26091320.234224962628100
4.0376-4.62130.231300.20862509263999
4.6213-5.82030.22921330.20562515264898
5.8203-44.45870.24391400.22862641278199
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)
13.0649-0.58240.77153.65950.14473.2844-0.02780.01730.1215-0.0609-0.1592-0.60620.2268-0.0099-0.00030.70540.03440.02980.90760.11070.6581-38.0279-87.1423-4.4215
22.92610.82331.12036.4676-0.75413.6324-0.294-0.390.7603-0.1229-0.06680.5343-0.9218-0.4713-0.00291.05150.2311-0.15280.9695-0.15020.9472-45.3211-55.96710.8897
33.917-0.1739-1.32333.4282-1.1392.26060.1724-0.4740.27110.2288-0.1192-0.8014-0.06980.4501-0.00060.9719-0.0792-0.4531.1822-0.01841.4988-14.453-65.17675.9221
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1(chain 'C' and resid 24 through 175)C24 - 175
2X-RAY DIFFRACTION2(chain 'B' and resid 30 through 175)B30 - 175
3X-RAY DIFFRACTION3(chain 'A' and resid 31 through 175)A31 - 175

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