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- EMDB-29934: Cryo-EM structure of hAQP2 in DDM -

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Basic information

Entry
Database: EMDB / ID: EMD-29934
TitleCryo-EM structure of hAQP2 in DDM
Map data
Sample
  • Complex: tetrameter of hAQP2
    • Protein or peptide: Aquaporin-2
Keywordschannel / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsKamegawa A / Suzuki S / Nishikawa K / Numoto N / Suzuki H / Fujiyoshi Y
Funding support Japan, 1 items
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, 2023-
Header (metadata) releaseJun 21, 2023-
Map releaseJun 21, 2023-
UpdateJul 5, 2023-
Current statusJul 5, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29934.png

Downloads & links

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Map

FileDownload / File: emd_29934.map.gz / Format: CCP4 / Size: 4.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.96 Å
Density
Contour LevelBy AUTHOR: 0.44
Minimum - Maximum-1.7826412 - 2.68054
Average (Standard dev.)0.05337124 (±0.21276847)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions104104104
Spacing104104104
CellA=B=C: 99.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_29934_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29934_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_29934_half_map_2.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 : tetrameter of hAQP2

EntireName: tetrameter of hAQP2
Components
  • Complex: tetrameter of hAQP2
    • Protein or peptide: Aquaporin-2

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Supramolecule #1: tetrameter of hAQP2

SupramoleculeName: tetrameter of hAQP2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Aquaporin-2

MacromoleculeName: Aquaporin-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 28.862389 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MWELRSIAFS RAVFAEFLAT LLFVFFGLGS ALNWPQALPS VLQIAMAFGL GIGTLVQALG HISGAHINPA VTVACLVGCH VSVLRAAFY VAAQLLGAVA GAALLHEITP ADIRGDLAVN ALSNSTTAGQ AVTVELFLTL QLVLCIFAST DERRGENPGT P ALSIGFSV ...String:
MWELRSIAFS RAVFAEFLAT LLFVFFGLGS ALNWPQALPS VLQIAMAFGL GIGTLVQALG HISGAHINPA VTVACLVGCH VSVLRAAFY VAAQLLGAVA GAALLHEITP ADIRGDLAVN ALSNSTTAGQ AVTVELFLTL QLVLCIFAST DERRGENPGT P ALSIGFSV ALGHLLGIHY TGCSMNPARS LAPAVVTGKF DDHWVFWIGP LVGAILGSLL YNYVLFPPAK SLSERLAVLK GL EPDTDWE EREVRRRQSV ELHSPQSLPR GTKA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration7.2 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 69.6 e/Å2

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Image processing

Startup modelType of model: OTHER
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 236700
FSC plot (resolution estimation)

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