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 transport / water channel activity ...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 transport / water channel activity / 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 / basolateral plasma membrane / protein homotetramerization / 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
Journal: 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.
Idetical with deposited unit in distinct coordinate
point asymmetric unit, std point frame
Type
Name
Symmetry operation
Number
transform to point frame
1
Symmetry
Point symmetry: (Schoenflies symbol: C4 (4 fold cyclic))
Noncrystallographic symmetry (NCS)
NCS oper:
ID
Code
Matrix
Vector
1
given
(1), (1), (1)
2
generate
(-1), (1), (1)
99.84
3
generate
(-1), (-1), (1)
99.84, 99.84
4
generate
(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
Experiment
Method: ELECTRON MICROSCOPY
EM experiment
Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction
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Sample preparation
Component
Name: tetrameter of hAQP2 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weight
Experimental value: NO
Source (natural)
Organism: Homo sapiens (human)
Source (recombinant)
Organism: Spodoptera frugiperda (fall armyworm)
Buffer solution
pH: 8
Specimen
Conc.: 7.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Resolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 236700 / Symmetry type: POINT
Refinement
Resolution: 2.89→2.89 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