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- PDB-8gcl: Cryo-EM structure of hAQP2 in DDM -

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Basic information

Entry
Database: PDB / ID: 8gcl
TitleCryo-EM structure of hAQP2 in DDM
ComponentsAquaporin-2
KeywordsMEMBRANE PROTEIN / channel
Function / homology
Function and homology information


cellular response to water deprivation / renal water transport / glycerol transmembrane transporter activity / Passive transport by Aquaporins / glycerol transmembrane transport / lumenal side of membrane / water transmembrane transporter activity / cellular response to mercury ion / water channel activity / water transport ...cellular response to water deprivation / renal water transport / glycerol transmembrane transporter activity / Passive transport by Aquaporins / glycerol transmembrane transport / lumenal side of membrane / water transmembrane transporter activity / cellular response to mercury ion / water channel activity / water transport / metanephric collecting duct development / renal water homeostasis / transport vesicle membrane / cellular response to copper ion / actin filament organization / recycling endosome / Vasopressin regulates renal water homeostasis via Aquaporins / protein homotetramerization / basolateral plasma membrane / apical plasma membrane / perinuclear region of cytoplasm / Golgi apparatus / extracellular exosome / membrane / 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
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsKamegawa, A. / Suzuki, S. / Nishikawa, K. / Numoto, N. / Suzuki, H. / Fujiyoshi, Y.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H00451 Japan
CitationJournal: J Struct Biol / Year: 2023
Title: Structural analysis of the water channel AQP2 by single-particle cryo-EM.
Authors: Akiko Kamegawa / Shota Suzuki / Hiroshi Suzuki / Kouki Nishikawa / Nobutaka Numoto / Yoshinori Fujiyoshi /
Abstract: Water channels, which are small membrane proteins almost entirely buried in lipid membranes, are challenging research targets for single-particle cryo-electron microscopy (cryo-EM), a powerful ...Water channels, which are small membrane proteins almost entirely buried in lipid membranes, are challenging research targets for single-particle cryo-electron microscopy (cryo-EM), a powerful technique routinely used to determine the structures of membrane proteins. Because the single-particle method enables structural analysis of a whole protein with flexible parts that interfere with crystallization, we have focused our efforts on analyzing water channel structures. Here, utilizing this system, we analyzed the structure of full-length aquaporin-2 (AQP2), a primary regulator of vasopressin-dependent reabsorption of water at the renal collecting ducts. The 2.9 Å resolution map revealed a cytoplasmic extension of the cryo-EM density that was presumed to be the highly flexible C-terminus at which the localization of AQP2 is regulated in the renal collecting duct cells. We also observed a continuous density along the common water pathway inside the channel pore and lipid-like molecules at the membrane interface. Observations of these constructions in the AQP2 structure analyzed without any fiducial markers (e.g., a rigidly bound antibody) indicate that single-particle cryo-EM will be useful for investigating water channels in native states as well as in complexes with chemical compounds.
History
DepositionMar 2, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 21, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 5, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Aquaporin-2


Theoretical massNumber of molelcules
Total (without water)28,8621
Polymers28,8621
Non-polymers00
Water0
1
A: Aquaporin-2

A: Aquaporin-2

A: Aquaporin-2

A: Aquaporin-2


Theoretical massNumber of molelcules
Total (without water)115,4504
Polymers115,4504
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation3
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C4 (4 fold cyclic))
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-1), (1), (1)99.84
3generate(-1), (-1), (1)99.84, 99.84
4generate(1), (-1), (1)99.84

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Components

#1: Protein Aquaporin-2 / / AQP-2 / ADH water channel / Aquaporin-CD / AQP-CD / Collecting duct water channel protein / WCH-CD ...AQP-2 / ADH water channel / Aquaporin-CD / AQP-CD / Collecting duct water channel protein / WCH-CD / Water channel protein for renal collecting duct


Mass: 28862.389 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: AQP2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P41181

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: tetrameter of hAQP2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenConc.: 7.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: MOLYBDENUM / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 69.6 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0352 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 236700 / Symmetry type: POINT
RefinementResolution: 2.9→94.08 Å / Cor.coef. Fo:Fc: 0.838 / SU B: 11.101 / SU ML: 0.207 / ESU R: 0.194
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.35629 --
obs0.35629 56865 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 91.506 Å2
Refinement stepCycle: 1 / Total: 1754
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0080.0111795
ELECTRON MICROSCOPYr_bond_other_d00.0161714
ELECTRON MICROSCOPYr_angle_refined_deg1.2421.6052455
ELECTRON MICROSCOPYr_angle_other_deg0.391.5353948
ELECTRON MICROSCOPYr_dihedral_angle_1_deg3.545238
ELECTRON MICROSCOPYr_dihedral_angle_2_deg6.751107
ELECTRON MICROSCOPYr_dihedral_angle_3_deg17.00310259
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.0610.2300
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.022042
ELECTRON MICROSCOPYr_gen_planes_other0.0010.02366
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it11.4718.857955
ELECTRON MICROSCOPYr_mcbond_other11.4718.857955
ELECTRON MICROSCOPYr_mcangle_it17.12713.3441192
ELECTRON MICROSCOPYr_mcangle_other17.11913.381193
ELECTRON MICROSCOPYr_scbond_it11.3879.698840
ELECTRON MICROSCOPYr_scbond_other11.3819.73841
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other17.09814.131264
ELECTRON MICROSCOPYr_long_range_B_refined25.3577762
ELECTRON MICROSCOPYr_long_range_B_other25.3557763
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.9→2.975 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.359 4182 -
obs--100 %

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