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

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Basic information

Entry
Database: PDB / ID: 8sjx
TitleStructure of lens aquaporin-0 array in sphingomyelin/cholesterol bilayer (2SM:1Chol)
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 / positive regulation of cell adhesion / visual perception / protein homotetramerization / calmodulin binding ...gap junction-mediated intercellular transport / water channel activity / water transport / structural constituent of eye lens / gap junction / lens development in camera-type eye / 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
Citation
Journal: Elife / Year: 2024
Title: Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts.
Authors: Po-Lin Chiu / Juan D Orjuela / Bert L de Groot / Camilo Aponte Santamaría / Thomas Walz /
Abstract: Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron ...Aquaporin-0 (AQP0) tetramers form square arrays in lens membranes through a yet unknown mechanism, but lens membranes are enriched in sphingomyelin and cholesterol. Here, we determined electron crystallographic structures of AQP0 in sphingomyelin/cholesterol membranes and performed molecular dynamics (MD) simulations to establish that the observed cholesterol positions represent those seen around an isolated AQP0 tetramer and that the AQP0 tetramer largely defines the location and orientation of most of its associated cholesterol molecules. At a high concentration, cholesterol increases the hydrophobic thickness of the annular lipid shell around AQP0 tetramers, which may thus cluster to mitigate the resulting hydrophobic mismatch. Moreover, neighboring AQP0 tetramers sandwich a cholesterol deep in the center of the membrane. MD simulations show that the association of two AQP0 tetramers is necessary to maintain the deep cholesterol in its position and that the deep cholesterol increases the force required to laterally detach two AQP0 tetramers, not only due to protein-protein contacts but also due to increased lipid-protein complementarity. Since each tetramer interacts with four such 'glue' cholesterols, avidity effects may stabilize larger arrays. The principles proposed to drive AQP0 array formation could also underlie protein clustering in lipid rafts.
#1: Journal: Elife / Year: 2024
Title: Structure and dynamics of cholesterol-mediated aquaporin-0 arrays and implications for lipid rafts
Authors: Chiu, P.L. / Orjuela, J.D. / de Groot, B.L. / Aponte-Santamaria, C. / Walz, T.
History
DepositionApr 18, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 24, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Database references / Structure summary / Category: citation / citation_author / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _pdbx_entry_details.has_protein_modification
Revision 1.2Nov 13, 2024Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lens fiber major intrinsic protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)33,5939
Polymers28,2851
Non-polymers5,3088
Water19811
1
A: Lens fiber major intrinsic protein
hetero molecules
x 8


Theoretical massNumber of molelcules
Total (without water)268,74272
Polymers226,2798
Non-polymers42,46364
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
Buried area40060 Å2
ΔGint-314 kcal/mol
Surface area70190 Å2
MethodPISA
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) / 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.: 7 / Source method: obtained synthetically / Formula: C39H79N2O6P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


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

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

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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 2:1 molar ratio.
Lipid protein ratio: 0.2 / Temperature: 310 K / Time: 7 DAY
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMMESMES1
2300 mMSodium chlorideNaCl1
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: 214
EM diffraction shellResolution: 2.3→13.8 Å / Fourier space coverage: 86.71 % / Multiplicity: 6.2 / Num. of structure factors: 16437 / Phase residual: 1.0E-5 °
EM diffraction statsDetails: There was no phase error rejection criteria used for diffraction intensities.
Fourier space coverage: 86.71 % / High resolution: 2.3 Å / Num. of intensities measured: 122501 / Num. of structure factors: 16437 / Phase error rejection criteria: 0 / Rmerge: 21.6 / Rsym: 14.9
ReflectionBiso Wilson estimate: 49.13 Å2

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
PHASERphasing
IPLT0.9.8data reduction
EM software
IDNameVersionCategoryDetails
1DigitalMicrograph2image acquisitionLow dose imaging
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∠γ: 90 ° / C sampling length: 200 Å / A: 65.5 Å / B: 65.5 Å / C: 200 Å / Space group name H-M: P422
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: 0.3971 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 33.7767
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflectionSelection details
Rfree0.2863 1643 10 %Random selection
Rwork0.2595 14794 --
obs0.2622 16437 86.71 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 66.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00842045
ELECTRON CRYSTALLOGRAPHYf_angle_d1.09192749
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0395294
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0054307
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d18.3187434
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.35-2.420.43691310.42351174ELECTRON CRYSTALLOGRAPHY84.63
2.42-2.50.4031310.38131188ELECTRON CRYSTALLOGRAPHY86.1
2.5-2.580.42581300.41121169ELECTRON CRYSTALLOGRAPHY85.4
2.59-2.690.36021330.36181201ELECTRON CRYSTALLOGRAPHY86.12
2.69-2.810.40321360.3591225ELECTRON CRYSTALLOGRAPHY86.8
2.81-2.960.37761340.34911214ELECTRON CRYSTALLOGRAPHY86.91
2.96-3.140.34491370.30421227ELECTRON CRYSTALLOGRAPHY86.99
3.14-3.380.26451370.24921225ELECTRON CRYSTALLOGRAPHY87.48
3.38-3.710.24351380.23951256ELECTRON CRYSTALLOGRAPHY88.01
3.71-4.240.22171440.20721285ELECTRON CRYSTALLOGRAPHY89.15
4.24-5.280.24991450.2111312ELECTRON CRYSTALLOGRAPHY89.44
5.29-13.750.27191470.23231318ELECTRON CRYSTALLOGRAPHY83.91

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