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- EMDB-5679: Electron Microscopy of the Aquaporin-0/Calmodulin Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-5679
TitleElectron Microscopy of the Aquaporin-0/Calmodulin Complex
Map data3D Reconstruction of the Aquaporin-0/Calmodulin Complex
Sample
  • Sample: Aquaporin-0 bound to Calmodulin
  • Protein or peptide: Aquaporin-0
  • Protein or peptide: Calmodulin
Keywordsaquaporin / calmodulin / calcium regulation / water channel / membrane protein complex / electron microscopy
Function / homology
Function and homology information


maintenance of lens transparency / cell adhesion mediator activity / homotypic cell-cell adhesion / gap junction-mediated intercellular transport / water transport / water channel activity / : / : / : / : ...maintenance of lens transparency / cell adhesion mediator activity / homotypic cell-cell adhesion / gap junction-mediated intercellular transport / water transport / water channel activity / : / : / : / : / : / positive regulation of protein autophosphorylation / structural constituent of eye lens / negative regulation of peptidyl-threonine phosphorylation / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / lens development in camera-type eye / CaM pathway / positive regulation of peptidyl-threonine phosphorylation / Cam-PDE 1 activation / Sodium/Calcium exchangers / anchoring junction / Calmodulin induced events / Reduction of cytosolic Ca++ levels / positive regulation of DNA binding / Activation of Ca-permeable Kainate Receptor / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Loss of phosphorylation of MECP2 at T308 / CREB1 phosphorylation through the activation of Adenylate Cyclase / CaMK IV-mediated phosphorylation of CREB / PKA activation / response to corticosterone / negative regulation of high voltage-gated calcium channel activity / Glycogen breakdown (glycogenolysis) / CLEC7A (Dectin-1) induces NFAT activation / Activation of RAC1 downstream of NMDARs / negative regulation of ryanodine-sensitive calcium-release channel activity / organelle localization by membrane tethering / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / negative regulation of calcium ion export across plasma membrane / regulation of cardiac muscle cell action potential / presynaptic endocytosis / nitric-oxide synthase binding / Synthesis of IP3 and IP4 in the cytosol / regulation of cell communication by electrical coupling involved in cardiac conduction / regulation of synaptic vesicle exocytosis / Phase 0 - rapid depolarisation / calcineurin-mediated signaling / Negative regulation of NMDA receptor-mediated neuronal transmission / Unblocking of NMDA receptors, glutamate binding and activation / RHO GTPases activate PAKs / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / adenylate cyclase binding / regulation of ryanodine-sensitive calcium-release channel activity / protein phosphatase activator activity / Long-term potentiation / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / positive regulation of protein serine/threonine kinase activity / DARPP-32 events / catalytic complex / Smooth Muscle Contraction / detection of calcium ion / regulation of synaptic vesicle endocytosis / regulation of cardiac muscle contraction / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / activation of adenylate cyclase activity / cellular response to interferon-beta / Protein methylation / phosphatidylinositol 3-kinase binding / calcium channel inhibitor activity / Activation of AMPK downstream of NMDARs / presynaptic cytosol / positive regulation of nitric-oxide synthase activity / Ion homeostasis / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / enzyme regulator activity / eNOS activation / titin binding / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / sperm midpiece / regulation of calcium-mediated signaling / voltage-gated potassium channel complex / calcium channel complex / visual perception / FCERI mediated Ca+2 mobilization / substantia nigra development / Ras activation upon Ca2+ influx through NMDA receptor / regulation of heart rate / FCGR3A-mediated IL10 synthesis / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / calyx of Held / response to amphetamine / adenylate cyclase activator activity / sarcomere / VEGFR2 mediated cell proliferation / protein serine/threonine kinase activator activity
Similarity search - Function
Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / Major intrinsic protein / Major intrinsic protein / Major intrinsic protein / Aquaporin-like / : / EF-hand domain pair / EF-hand, calcium binding motif ...Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / Major intrinsic protein / Major intrinsic protein / Major intrinsic protein / Aquaporin-like / : / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site / EF-hand calcium-binding domain. / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Calmodulin-1 / Calmodulin-3 / Pas12 / Lens fiber major intrinsic protein
Similarity search - Component
Biological speciesOvis aries (sheep) / Homo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsReichow SL / Clemens DM / Freites JA / Nemeth-Cahalan KL / Heyden M / Tobias DJ / Hall JE / Gonen T
CitationJournal: Nat Struct Mol Biol / Year: 2013
Title: Allosteric mechanism of water-channel gating by Ca2+-calmodulin.
Authors: Steve L Reichow / Daniel M Clemens / J Alfredo Freites / Karin L Németh-Cahalan / Matthias Heyden / Douglas J Tobias / James E Hall / Tamir Gonen /
Abstract: Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is ...Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudoatomic structure of full-length mammalian aquaporin-0 (AQP0, Bos taurus) in complex with CaM, using EM to elucidate how this signaling protein modulates water-channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.
History
DepositionMay 30, 2013-
Header (metadata) releaseJun 26, 2013-
Map releaseAug 14, 2013-
UpdateSep 18, 2013-
Current statusSep 18, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.96
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 4.96
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j41
  • Surface level: 4.96
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5679.tif

Downloads & links

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Map

FileDownload / File: emd_5679.map.gz / Format: CCP4 / Size: 1.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D Reconstruction of the Aquaporin-0/Calmodulin Complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.98 Å/pix.
x 66 pix.
= 262.68 Å		3.98 Å/pix.
x 66 pix.
= 262.68 Å		3.98 Å/pix.
x 66 pix.
= 262.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.98 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.96 / Movie #1: 4.96
Minimum - Maximum-3.1143434 - 8.37446785
Average (Standard dev.)-0.00000001 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-33-33-33
Dimensions666666
Spacing666666
CellA=B=C: 262.68 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.983.983.98
M x/y/z666666
origin x/y/z0.0000.0000.000
length x/y/z262.680262.680262.680
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-33-33-33
NC/NR/NS666666
D min/max/mean-3.1148.374-0.000

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Supplemental data

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Sample components

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Entire : Aquaporin-0 bound to Calmodulin

EntireName: Aquaporin-0 bound to Calmodulin
Components
  • Sample: Aquaporin-0 bound to Calmodulin
  • Protein or peptide: Aquaporin-0
  • Protein or peptide: Calmodulin

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Supramolecule #1000: Aquaporin-0 bound to Calmodulin

SupramoleculeName: Aquaporin-0 bound to Calmodulin / type: sample / ID: 1000
Details: Sample was prepared for electron microscopy with negative stain
Oligomeric state: One tetramer of Aquaporin-0 bound to 2 molecules of Calmodulin
Number unique components: 2
Molecular weightExperimental: 130 KDa / Theoretical: 130 KDa / Method: Size-exclusion Chromatography and SDS-PAGE

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Macromolecule #1: Aquaporin-0

MacromoleculeName: Aquaporin-0 / type: protein_or_peptide / ID: 1 / Name.synonym: AQP0, MIP / Details: Crosslinked to Calmodulin using EDC/NHS / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Ovis aries (sheep) / synonym: Sheep / Tissue: eye lens / Cell: fiber cell / Location in cell: plasma membrane
Molecular weightExperimental: 25 KDa / Theoretical: 25 KDa
SequenceUniProtKB: Pas12 / InterPro: Major intrinsic protein

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Macromolecule #2: Calmodulin

MacromoleculeName: Calmodulin / type: protein_or_peptide / ID: 2 / Name.synonym: CaM / Details: Calmodulin crosslinked to Aquaporin-0 / Number of copies: 2 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: cytoplasmic
Molecular weightExperimental: 17 KDa / Theoretical: 17 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 / Recombinant plasmid: pET
SequenceUniProtKB: Calmodulin-3

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 7.4 / Details: 25mM HEPES, 5mM CaCl2, 0.3% decylmaltoside
StainingType: NEGATIVE / Details: 0.75% uranyl formate
GridDetails: 400 mesh carbon coated grid (Ted Pella)
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 12
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Legacy - Electron beam tilt params: 0
DateFeb 25, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 200 / Average electron dose: 15 e/Å2 / Bits/pixel: 16
Tilt angle min0
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: OTHER / Tilt angle max: 50

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Image processing

DetailsParticles were selected from a tilted pair dataset at 0 and 50 degree tilt using SPIDER. An initial reconstruction was generated using random conical tilt methods in SPIDER and refined in FREALIGN
CTF correctionDetails: CTF-TILT, each micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER, FREALIGN
Details: Final Map with C2 Symmetry and Filtered to 25 Angstrom
Number images used: 11720

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Atomic model buiding 1

Initial modelPDB ID:

2b6p


Chain - Chain ID: A
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j41:
Pseudo-atomic model of the Aquaporin-0/Calmodulin complex derived from electron microscopy

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