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- PDB-8sjy: Structure of lens aquaporin-0 array in sphingomyelin/cholesterol ... -

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Basic information

Entry
Database: PDB / ID: 8sjy
TitleStructure of lens aquaporin-0 array in sphingomyelin/cholesterol bilayer (1SM:2Chol)
ComponentsLens fiber major intrinsic protein
KeywordsMEMBRANE PROTEIN / aquaporin / lens / cholesterol / lipid raft
Function / homology
Function and homology information


gap junction-mediated intercellular transport / water channel activity / water transport / structural constituent of eye lens / gap junction / lens development in camera-type eye / response to stimulus / positive regulation of cell adhesion / visual perception / protein homotetramerization ...gap junction-mediated intercellular transport / water channel activity / water transport / structural constituent of eye lens / gap junction / lens development in camera-type eye / response to stimulus / positive regulation of cell adhesion / visual perception / protein homotetramerization / calmodulin binding / endoplasmic reticulum / plasma membrane
Similarity search - Function
Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / Major intrinsic protein / Major intrinsic protein / Aquaporin-like
Similarity search - Domain/homology
CHOLESTEROL / Chem-HWP / Lens fiber major intrinsic protein
Similarity search - Component
Biological speciesOvis aries (sheep)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / Resolution: 2.5 Å
AuthorsChiu, P.-L. / Walz, T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)EY015107 United States
CitationJournal: To Be Published
Title: Structure of aquaporin-0 arrays in sphingomyelin/cholesterol membranes and implications for lipid rafts
Authors: Chiu, P.-L. / Walz, T.
History
DepositionApr 18, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lens fiber major intrinsic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,34710
Polymers28,2851
Non-polymers5,0629
Water19811
1
A: Lens fiber major intrinsic protein
hetero molecules

A: Lens fiber major intrinsic protein
hetero molecules

A: Lens fiber major intrinsic protein
hetero molecules

A: Lens fiber major intrinsic protein
hetero molecules

A: Lens fiber major intrinsic protein
hetero molecules

A: Lens fiber major intrinsic protein
hetero molecules

A: Lens fiber major intrinsic protein
hetero molecules

A: Lens fiber major intrinsic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)266,77380
Polymers226,2798
Non-polymers40,49472
Water1448
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_755-x+2,-y,z1
crystal symmetry operation3_645-y+1,x-1,z1
crystal symmetry operation4_665y+1,-x+1,z1
crystal symmetry operation5_755-x+2,y,-z1
crystal symmetry operation6_555x,-y,-z1
crystal symmetry operation7_645y+1,x-1,-z1
crystal symmetry operation8_665-y+1,-x+1,-z1
Unit cell
Length a, b, c (Å)65.500, 65.500, 200.000
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number89
Space group name H-MP422
Space group name HallP42

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Components

#1: Protein Lens fiber major intrinsic protein / / Aquaporin-0


Mass: 28284.885 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Ovis aries (sheep) / Organ: Eye / Tissue: Lens / References: UniProt: Q6J8I9
#2: Chemical
ChemComp-HWP / [(E,2S,3R)-2-(hexadecanoylamino)-3-oxidanyl-octadec-4-enyl] 2-(trimethylazaniumyl)ethyl phosphate / N-Palmitoylsphingomyelin


Mass: 703.028 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C39H79N2O6P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CLR / CHOLESTEROL / Cholesterol


Mass: 386.654 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 11 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography
Crystal symmetry

Image processing-ID: 1 / ∠γ: 90 ° / C sampling length: 200 Å / A: 65.5 Å / B: 65.5 Å / C: 200 Å / Space group name H-M: P422

ID
1
2

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Sample preparation

ComponentName: Lens aquaporin-0 in sphingomyelin/cholesterol bilayer / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.0283 MDa / Experimental value: NO
Source (natural)Organism: Ovis aries (sheep) / Organ: Eye / Tissue: Lens
EM crystal formationLipid mixture: Sphingomyelin and cholesterol were mixed at a 1:2 molar ratio.
Lipid protein ratio: 0.2 / Temperature: 310 K / Time: 7 DAY
Buffer solutionpH: 6
Details: 10 mM MES (pH 6.0), 300 mM NaCl, 30 mM MgCl2, and 0.05% NaN3
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMMESMES1
2300 mMSodium chlorideNaClSodium chloride1
330 mMMagnesium chlorideMgCl21
40.05 %Sodium azideNaN31
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO / Details: 2D crystal of lens AQP0.
Specimen supportGrid material: MOLYBDENUM / Grid type: Homemade
EM embeddingDetails: Aquaporin-0 2D crystals were prepared on molybdenum grids using the carbon sandwich method and a trehalose concentration ranging from 3% to 5% (w/v).
Material: Trehalose

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Data collection

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Details: The diffraction patterns were recorded without setting defocus.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / Calibrated defocus min: 0 nm / Calibrated defocus max: 0 nm / C2 aperture diameter: 30 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 30 sec. / Electron dose: 10 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Num. of diffraction images: 241
EM diffraction shellResolution: 2.3→11.8 Å / Fourier space coverage: 90.19 % / Multiplicity: 6.3 / Num. of structure factors: 17031 / Phase residual: 1.0E-6 °
EM diffraction statsDetails: There was no phase error rejection criteria used for diffraction intensities.
Fourier space coverage: 90.19 % / High resolution: 2.3 Å / Num. of intensities measured: 127703 / Num. of structure factors: 17031 / Phase error rejection criteria: 0 / Rmerge: 19.9 / Rsym: 13.8
ReflectionBiso Wilson estimate: 31.17 Å2

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Processing

Software
NameVersionClassification
phenix.refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
PHASERphasing
IPLT0.9.8data reduction
EM software
IDNameVersionCategoryDetails
1DigitalMicrograph2image acquisition
8CCP4 package6molecular replacementPhaser 2.1
11IPET0.9.8crystallography merginghttps://iplt.biozentrum.unibas.ch/diff/introduction.html
12IPET0.9.83D reconstructionhttps://iplt.biozentrum.unibas.ch/diff/introduction.html
13CNS1.3model refinementModel refinement
14PHENIX1.20.1model refinementModel refinement
Crystal symmetry

Image processing-ID: 1 / ∠γ: 90 ° / C sampling length: 200 Å / A: 65.5 Å / B: 65.5 Å / C: 200 Å / Space group name H-M: P422

ID
1
2
CTF correctionType: NONE
3D reconstructionResolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 2D CRYSTAL
Atomic model buildingPDB-ID: 2B6O

2b6o


Pdb chain-ID: A / Accession code: 2B6O / Chain residue range: 6-255 / Details: Molecular replacement / Pdb chain residue range: 6-255 / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.5→2.5 Å / SU ML: 0.3818 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 32.4947
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.2866 1704 10.01 %Random selection
Rwork0.2617 15327 --
obs0.2642 17031 90.19 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 51.15 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00532062
ELECTRON CRYSTALLOGRAPHYf_angle_d0.83812801
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0374314
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0046303
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d15.071417
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.35-2.420.43041340.41781201ELECTRON CRYSTALLOGRAPHY86.86
2.42-2.50.40941360.38721225ELECTRON CRYSTALLOGRAPHY89.07
2.5-2.580.42981350.37871218ELECTRON CRYSTALLOGRAPHY88.72
2.58-2.690.38011390.34611253ELECTRON CRYSTALLOGRAPHY90.04
2.69-2.810.3661390.33981252ELECTRON CRYSTALLOGRAPHY89.51
2.81-2.950.34151390.33321253ELECTRON CRYSTALLOGRAPHY89.69
2.95-3.130.33031410.28881273ELECTRON CRYSTALLOGRAPHY90.7
3.13-3.370.28821420.24961266ELECTRON CRYSTALLOGRAPHY91.19
3.37-3.70.241450.2361307ELECTRON CRYSTALLOGRAPHY91.32
3.7-4.210.22941470.19851324ELECTRON CRYSTALLOGRAPHY91.77
4.22-5.230.21661500.19421348ELECTRON CRYSTALLOGRAPHY92.53
5.23-11.760.24061570.22851407ELECTRON CRYSTALLOGRAPHY90.61

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