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- EMDB-5803: Electron crystallography of AQP0 with anionic phospholipids. -

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Basic information

Entry
Database: EMDB / ID: EMD-5803
TitleElectron crystallography of AQP0 with anionic phospholipids.
Map data3D reconstruction of AQP0_DMPG 2D crystal in p121 form
Sample
  • Sample: 2D crystal structure of AQP0 (Ovis aries) with DMPG lipid in p121 form
  • Protein or peptide: Aquaporin-0
Keywordsaquaporin-0 / AQP0 / DMPG / phosphatidylglycerol / DMPS / phosphatidylserine / anionic phospholipids / electron crystallography
Function / homology
Function and homology information


gap junction-mediated intercellular transport / water channel activity / water transport / structural constituent of eye lens / gap junction / response to stimulus / lens development in camera-type eye / 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 / response to stimulus / 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
Lens fiber major intrinsic protein
Similarity search - Component
Biological speciesOvis aries (sheep)
Methodelectron crystallography / Resolution: 8.0 Å
AuthorsHite RK / Chiu P-L / Schuller J / Walz T
CitationJournal: PLoS One / Year: 2015
Title: Effect of lipid head groups on double-layered two-dimensional crystals formed by aquaporin-0.
Authors: Richard Kevin Hite / Po-Lin Chiu / Jan Michael Schuller / Thomas Walz /
Abstract: Aquaporin-0 (AQP0) is a lens-specific water channel that also forms membrane junctions. Reconstitution of AQP0 with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL) yielded well- ...Aquaporin-0 (AQP0) is a lens-specific water channel that also forms membrane junctions. Reconstitution of AQP0 with dimyristoyl phosphatidylcholine (DMPC) and E. coli polar lipids (EPL) yielded well-ordered, double-layered two-dimensional (2D) crystals that allowed electron crystallographic structure determination of the AQP0-mediated membrane junction. The interacting tetramers in the two crystalline layers are exactly in register, resulting in crystals with p422 symmetry. The high-resolution density maps also allowed modeling of the annular lipids surrounding the tetramers. Comparison of the DMPC and EPL bilayers suggested that the lipid head groups do not play an important role in the interaction of annular lipids with AQP0. We now reconstituted AQP0 with the anionic lipid dimyristoyl phosphatidylglycerol (DMPG), which yielded a mixture of 2D crystals with different symmetries. The different crystal symmetries result from shifts between the two crystalline layers, suggesting that the negatively charged PG head group destabilizes the interaction between the extracellular AQP0 surfaces. Reconstitution of AQP0 with dimyristoyl phosphatidylserine (DMPS), another anionic lipid, yielded crystals that had the usual p422 symmetry, but the crystals showed a pH-dependent tendency to stack through their cytoplasmic surfaces. Finally, AQP0 failed to reconstitute into membranes that were composed of more than 40% dimyristoyl phosphatidic acid (DMPA). Hence, although DMPG, DMPS, and DMPA are all negatively charged lipids, they have very different effects on AQP0 2D crystals, illustrating the importance of the specific lipid head group chemistry beyond its mere charge.
History
DepositionNov 26, 2013-
Header (metadata) releaseMar 19, 2014-
Map releaseDec 10, 2014-
UpdateDec 10, 2014-
Current statusDec 10, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 70
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 70
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5803.gif

Downloads & links

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Map

FileDownload / File: emd_5803.map.gz / Format: CCP4 / Size: 120.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of AQP0_DMPG 2D crystal in p121 form
Voxel sizeX: 0.3275 Å / Y: 0.3275 Å / Z: 1 Å
Density
Contour LevelBy EMDB: 78.0 / Movie #1: 70
Minimum - Maximum-173.022537230000012 - 250.0
Average (Standard dev.)0.01140478 (±35.810249329999998)
SymmetrySpace group: 4
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-200-200-100
Dimensions401401201
Spacing200200200
CellA: 65.5 Å / B: 65.5 Å / C: 200.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.32750.32751
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z65.50065.500200.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-95-75153
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-200-200-100
NC/NR/NS401401201
D min/max/mean-173.023250.0000.011

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Supplemental data

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Sample components

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Entire : 2D crystal structure of AQP0 (Ovis aries) with DMPG lipid in p121 form

EntireName: 2D crystal structure of AQP0 (Ovis aries) with DMPG lipid in p121 form
Components
  • Sample: 2D crystal structure of AQP0 (Ovis aries) with DMPG lipid in p121 form
  • Protein or peptide: Aquaporin-0

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Supramolecule #1000: 2D crystal structure of AQP0 (Ovis aries) with DMPG lipid in p121 form

SupramoleculeName: 2D crystal structure of AQP0 (Ovis aries) with DMPG lipid in p121 form
type: sample / ID: 1000 / Details: 2D crystals / Oligomeric state: Tetramer / Number unique components: 1
Molecular weightTheoretical: 113 KDa

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Macromolecule #1: Aquaporin-0

MacromoleculeName: Aquaporin-0 / type: protein_or_peptide / ID: 1 / Name.synonym: AQP0
Details: Aquaporin-0 was crystallized with dimyristoylglycerol (DMPG) in p121 symmetry.
Number of copies: 32 / Oligomeric state: Tetramer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Ovis aries (sheep) / synonym: Sheep / Tissue: eye / Cell: Lens fiber cells / Organelle: Membrane / Location in cell: Plasma membranes
SequenceUniProtKB: Lens fiber major intrinsic protein

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Experimental details

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Structure determination

Processingelectron crystallography
Aggregation state2D array

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Sample preparation

BufferpH: 6 / Details: 10 mM MES, 50 mM Mg2Cl2, 150 mM NaCl, 0.05% NaN3
GridDetails: Carbon sandwich method combined with trehalose embedding
VitrificationCryogen name: NONE / Instrument: OTHER
DetailsDialysis.
Crystal formationDetails: Dialysis.

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm / Nominal defocus max: 0.65 µm / Nominal defocus min: 0.35 µm / Nominal magnification: 52000
Sample stageSpecimen holder: liquid-nitrogen cooled / Specimen holder model: OTHER
TemperatureMin: 90 K / Max: 100 K / Average: 95 K
DateMar 31, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Sampling interval: 15 µm / Number real images: 146 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit cell - A: 65.5 Å / Unit cell - B: 65.5 Å / Unit cell - C: 200 Å / Unit cell - γ: 90.0 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 1 21
CTF correctionDetails: Phase flipping with each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.0 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: 2DX, and, MRC / Details: The reconstruction map shows in 2x2 unit cells.
DetailsCrystal images were unbent and reconstructed using 2DX and MRC software.

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Atomic model buiding 1

Initial modelPDB ID:

2b6o


Chain - Chain ID: A
SoftwareName: UCSF Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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