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- PDB-3iyz: Structure of Aquaporin-4 S180D mutant at 10.0 A resolution from e... -

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Basic information

Entry
Database: PDB / ID: 3iyz
TitleStructure of Aquaporin-4 S180D mutant at 10.0 A resolution from electron micrograph
ComponentsAquaporin-4
KeywordsTRANSPORT PROTEIN / WATER TRANSPORT / WATER CHANNEL / AQUAPORIN / TWO-DIMENSIONAL CRYSTAL / MEMBRANE PROTEIN / BACULOVIRUS EXPRESSION SYSTEM / GLYCOPROTEIN / MEMBRANE / PHOSPHOPROTEIN / TRANSMEMBRANE / TRANSPORT
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 / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 10 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
DepositionJul 24, 2010Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 25, 2010Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Jul 18, 2018Group: Author supporting evidence / Data collection
Category: em_image_scans / em_single_particle_entity / em_software
Item: _em_software.image_processing_id
Revision 1.3Sep 6, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_database_related / pdbx_initial_refinement_model / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_database_related.content_type / _struct_ref_seq_dif.details

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Assembly

Deposited unit
A: Aquaporin-4


Theoretical massNumber of molelcules
Total (without water)36,5951
Polymers36,5951
Non-polymers00
Water0
1
A: Aquaporin-4

A: Aquaporin-4

A: Aquaporin-4

A: Aquaporin-4


Theoretical massNumber of molelcules
Total (without water)146,3814
Polymers146,3814
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_565-x,-y+1,z1
crystal symmetry operation3_555-y+1/2,x+1/2,z1
crystal symmetry operation4_455y-1/2,-x+1/2,z1
Unit cell
Length a, b, c (Å)69.0, 69.0, 160.0
Angle α, β, γ (deg.)90, 90, 90
Int Tables number90
Space group name H-MP4212

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Components

#1: Protein Aquaporin-4 / / AQP-4 / WCH4 / Mercurial-insensitive water channel / MIWC / Coordinate model: Cα atoms only


Mass: 36595.137 Da / Num. of mol.: 1 / Fragment: UNP RESIDUES 23-323 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rattus norvegicus (Norway rat) / Gene: Aqp4 / Plasmid: PBLUEBACHIS2B / Cell line (production host): SF9 / Production host: SPODOPTERA FRUGIPERDA (fall armyworm) / References: UniProt: P47863

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY / Number of used crystals: 1
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: rat aquaporin-4 S180D mutant / Type: COMPLEX / Details: tetramer. The sample was embedded into lipid.
Molecular weightValue: 0.032 MDa / Experimental value: NO
Buffer solutionName: MES buffer / pH: 6
Details: 10mM MES, 75mM NaCl, 50mM MgCl2, 2mM DTT, 1% glycerol, 7% trehalose
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: 10mM MES, 75mM NaCl, 50mM MgCl2, 2mM DTT, 1% glycerol, 7% trehalose
Specimen supportDetails: The molybdenum grids were covered with solid carbon
VitrificationInstrument: REICHERT-JUNG PLUNGER / Cryogen name: NITROGEN / Chamber temperature: 277 K
Method: The grid was blotted with filter paper and plunged into liquid nitrogen.
Crystal growpH: 6 / Details: pH 6.00

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Data collection

MicroscopyModel: JEOL KYOTO-3000SFF
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 90000 X / Nominal defocus max: 3730 nm / Nominal defocus min: 430 nm / Cs: 1.6 mm
Astigmatism: bjective lens astigmatism was corrected at 400,000 times magnification
Camera length: 0 mm
Specimen holderSpecimen holder model: OTHER
Specimen holder type: Top entry liquid helium cooled cryo specimen holder
Temperature: 4.2 K / Tilt angle max: 60 ° / Tilt angle min: -60 °
Image recordingElectron dose: 38 e/Å2 / Film or detector model: GENERIC CCD / Details: Tietz 4K CCD
DiffractionMean temperature: 4.2 K
DetectorDate: Feb 4, 2008
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthRelative weight: 1

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Processing

SoftwareName: MRC / Classification: data scaling
EM software
IDNameCategory
1Situsmodel fitting
2MRC3D reconstruction
CTF correctionDetails: Each image
3D reconstructionMethod: 2D-crystals / Resolution: 10 Å / Resolution method: OTHER / Nominal pixel size: 1.67 Å / Magnification calibration: Each image / Details: MRC / Symmetry type: 2D CRYSTAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient / Details: REFINEMENT PROTOCOL--rigid body
Atomic model buildingPDB-ID: 2ZZ9

2zz9

RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2ZZ9

2zz9


Highest resolution: 10 Å
Refinement stepCycle: LAST / Highest resolution: 10 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms223 0 0 0 223

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