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- EMDB-5202: Influence of the cytoplasmic domains of aquaporin-4 on water cond... -

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Basic information

Entry
Database: EMDB / ID: EMD-5202
TitleInfluence of the cytoplasmic domains of aquaporin-4 on water conduction and array formation.
Map dataThis is an image of a surface rendered of Aquaporin-4
Sample
  • Sample: rat aquaporin-4 S180D mutant
  • Protein or peptide: aquaporin-4
KeywordsWater channel / Aquaporin / Cell adhesion
Function / homology
Function and homology information


Passive transport by Aquaporins / cerebrospinal fluid secretion / renal water absorption / regulation of vascular endothelial growth factor production / cerebrospinal fluid circulation / astrocyte end-foot / water channel activity / intracellular water homeostasis / water transport / negative regulation of cell adhesion molecule production ...Passive transport by Aquaporins / cerebrospinal fluid secretion / renal water absorption / regulation of vascular endothelial growth factor production / cerebrospinal fluid circulation / astrocyte end-foot / water channel activity / intracellular water homeostasis / water transport / negative regulation of cell adhesion molecule production / cell projection membrane / multicellular organismal-level water homeostasis / Vasopressin regulates renal water homeostasis via Aquaporins / cellular response to interleukin-6 / negative regulation of interleukin-1 beta production / negative regulation of interleukin-6 production / cellular response to interleukin-1 / response to glucocorticoid / T-tubule / basal plasma membrane / cellular response to estradiol stimulus / female pregnancy / establishment of localization in cell / cellular response to glucose stimulus / sensory perception of sound / sarcolemma / carbon dioxide transport / cell-cell adhesion / cellular response to type II interferon / cell-cell junction / protein homotetramerization / basolateral plasma membrane / endosome membrane / external side of plasma membrane / protein-containing complex / extracellular region / identical protein binding / plasma membrane / cytoplasm
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
Biological speciesRattus norvegicus (Norway rat)
Methodelectron crystallography / cryo EM / Resolution: 10.0 Å
AuthorsMitsuma T / Tani K / Hiroaki Y / Kamegawa A / Suzuki H / Hibino H / Kurachi Y / Fujiyoshi Y
CitationJournal: J Mol Biol / Year: 2010
Title: Influence of the cytoplasmic domains of aquaporin-4 on water conduction and array formation.
Authors: Tadanori Mitsuma / Kazutoshi Tani / Yoko Hiroaki / Akiko Kamegawa / Hiroshi Suzuki / Hiroshi Hibino / Yoshihisa Kurachi / Yoshinori Fujiyoshi /
Abstract: Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the ...Phosphorylation of Ser180 in cytoplasmic loop D has been shown to reduce the water permeability of aquaporin (AQP) 4, the predominant water channel in the brain. However, when the structure of the S180D mutant (AQP4M23S180D), which was generated to mimic phosphorylated Ser180, was determined to 2.8 Å resolution using electron diffraction patterns, it showed no significant differences from the structure of the wild-type channel. High-resolution density maps usually do not resolve protein regions that are only partially ordered, but these can sometimes be seen in lower-resolution density maps calculated from electron micrographs. We therefore used images of two-dimensional crystals and determined the structure of AQP4M23S180D at 10 A resolution. The features of the 10-A density map are consistent with those of the previously determined atomic model; in particular, there were no indications of any obstruction near the cytoplasmic pore entrance. In addition, water conductance measurements, both in vitro and in vivo, show the same water permeability for wild-type and mutant AQP4M23, suggesting that the S180D mutation neither reduces water conduction through a conformational change nor reduces water conduction by interacting with a protein that would obstruct the cytoplasmic channel entrance. Finally, the 10-A map shows a cytoplasmic density in between four adjacent tetramers that most likely represents the association of four N termini. This finding supports the critical role of the N terminus of AQP4 in the stabilization of orthogonal arrays, as well as their interference through lipid modification of cysteine residues in the longer N-terminal isoform.
History
DepositionJun 2, 2010-
Header (metadata) releaseJul 22, 2010-
Map releaseOct 4, 2010-
UpdateDec 26, 2012-
Current statusDec 26, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3iyz
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3iyz
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5202_1.png

Downloads & links

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Map

FileDownload / File: emd_5202.map.gz / Format: CCP4 / Size: 380.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an image of a surface rendered of Aquaporin-4
Voxel sizeX=Y=Z: 1.97 Å
Density
Contour LevelBy EMDB: 0.2 / Movie #1: 0.3
Minimum - Maximum-1.80102742 - 1.07438624
Average (Standard dev.)-0.0040511 (±0.30231997)
SymmetrySpace group: 90
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0-17-40
Dimensions353581
Spacing343480
CellA: 66.98 Å / B: 66.98 Å / C: 157.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.971.971.97
M x/y/z343480
origin x/y/z0.0000.0000.000
length x/y/z66.98066.980157.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-99-99-99
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-170-40
NC/NR/NS353581
D min/max/mean-1.8011.074-0.004

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Supplemental data

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Segmentation: the same as the volume map

Annotationthe same as the volume map
Fileemd_5202_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : rat aquaporin-4 S180D mutant

EntireName: rat aquaporin-4 S180D mutant
Components
  • Sample: rat aquaporin-4 S180D mutant
  • Protein or peptide: aquaporin-4

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Supramolecule #1000: rat aquaporin-4 S180D mutant

SupramoleculeName: rat aquaporin-4 S180D mutant / type: sample / ID: 1000 / Details: The sample was embedded into lipid. / Oligomeric state: tetramer / Number unique components: 8
Molecular weightTheoretical: 32 KDa

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Macromolecule #1: aquaporin-4

MacromoleculeName: aquaporin-4 / type: protein_or_peptide / ID: 1 / Name.synonym: AQP4 / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: Yes
Source (natural)Organism: Rattus norvegicus (Norway rat) / synonym: Rat / Location in cell: Plasma membrane
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pBlueBacHis2b
SequenceGO: water transport / InterPro: INTERPRO: IPR012269

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state2D array

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Sample preparation

BufferpH: 6
Details: 10mM MES, 75mM NaCl, 50mM MgCl2, 2mM DTT, 1% glycerol, 7% trehalose
GridDetails: molybdenum grid covered with a thin carbon film
VitrificationCryogen name: NITROGEN / Chamber temperature: 4.2 K / Instrument: REICHERT-JUNG PLUNGER / Details: Vitrification instrument: Reichert plunger
Method: The grid was blotted with filter paper and plunged into liquid nitrogen.
Detailsdialysis
Crystal formationDetails: dialysis

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Electron microscopy

MicroscopeJEOL KYOTO-3000SFF
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.6 mm / Nominal defocus max: 3.73 µm / Nominal defocus min: 0.43 µm / Nominal magnification: 90000
Sample stageSpecimen holder: Top entry liquid helium cooled cryo specimen holder
Specimen holder model: OTHER / Tilt angle min: -60 / Tilt angle max: 60 / Tilt series - Axis1 - Min angle: -60 ° / Tilt series - Axis1 - Max angle: 60 °
TemperatureAverage: 4.2 K
Alignment procedureLegacy - Astigmatism: bjective lens astigmatism was corrected at 400,000 times magnification
Image recordingCategory: FILM / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Digitization - Scanner: OTHER / Digitization - Sampling interval: 15 µm / Number real images: 246 / Average electron dose: 38 e/Å2 / Bits/pixel: 16

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Image processing

Crystal parametersUnit cell - A: 69.0 Å / Unit cell - B: 69.0 Å / Unit cell - C: 160.0 Å / Unit cell - γ: 90.0 ° / Unit cell - α: 90.0 ° / Unit cell - β: 90.0 ° / Plane group: P 4 21 2
CTF correctionDetails: Each image
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.0 Å / Resolution method: OTHER / Software - Name: MRC

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