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

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Basic information

Entry
Database: PDB / ID: 3j41
TitlePseudo-atomic model of the Aquaporin-0/Calmodulin complex derived from electron microscopy
Components
  • Calmodulin
  • Lens fiber major intrinsic protein
KeywordsTRANSPORT PROTEIN/CALCIUM BINDING / calcium regulation / water channel / membrane protein complex / TRANSPORT PROTEIN-CALCIUM BINDING complex
Function / homology
Function and homology information


maintenance of lens transparency / homotypic cell-cell adhesion / water channel activity / : / : / : / : / cell adhesion mediator activity / : / positive regulation of protein autophosphorylation ...maintenance of lens transparency / homotypic cell-cell adhesion / water channel activity / : / : / : / : / cell adhesion mediator activity / : / positive regulation of protein autophosphorylation / structural constituent of eye lens / water transport / negative regulation of peptidyl-threonine phosphorylation / : / type 3 metabotropic glutamate receptor binding / anchoring junction / positive regulation of peptidyl-threonine phosphorylation / positive regulation of DNA binding / CaM pathway / Cam-PDE 1 activation / Sodium/Calcium exchangers / Calmodulin induced events / Reduction of cytosolic Ca++ levels / 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 / PKA activation / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / response to corticosterone / negative regulation of ryanodine-sensitive calcium-release channel activity / Activation of RAC1 downstream of NMDARs / organelle localization by membrane tethering / CLEC7A (Dectin-1) induces NFAT activation / : / autophagosome membrane docking / negative regulation of high voltage-gated calcium channel activity / regulation of synaptic vesicle exocytosis / negative regulation of calcium ion export across plasma membrane / regulation of cardiac muscle cell action potential / presynaptic endocytosis / positive regulation of protein serine/threonine kinase activity / Synthesis of IP3 and IP4 in the cytosol / Phase 0 - rapid depolarisation / Negative regulation of NMDA receptor-mediated neuronal transmission / Unblocking of NMDA receptors, glutamate binding and activation / calcineurin-mediated signaling / nitric-oxide synthase binding / RHO GTPases activate PAKs / regulation of cell communication by electrical coupling involved in cardiac conduction / Ion transport by P-type ATPases / adenylate cyclase binding / Uptake and function of anthrax toxins / protein phosphatase activator activity / regulation of ryanodine-sensitive calcium-release channel activity / Long-term potentiation / Calcineurin activates NFAT / Regulation of MECP2 expression and activity / DARPP-32 events / regulation of synaptic vesicle endocytosis / Smooth Muscle Contraction / catalytic complex / detection of calcium ion / regulation of cardiac muscle contraction / cellular response to interferon-beta / RHO GTPases activate IQGAPs / phosphatidylinositol 3-kinase binding / activation of adenylate cyclase activity / calcium channel inhibitor activity / positive regulation of nitric-oxide synthase activity / presynaptic cytosol / enzyme regulator activity / Activation of AMPK downstream of NMDARs / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / eNOS activation / Ion homeostasis / regulation of calcium-mediated signaling / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Protein methylation / titin binding / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / voltage-gated potassium channel complex / FCERI mediated Ca+2 mobilization / calcium channel complex / substantia nigra development / regulation of heart rate / response to amphetamine / FCGR3A-mediated IL10 synthesis / calyx of Held / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / Ras activation upon Ca2+ influx through NMDA receptor / nitric-oxide synthase regulator activity / adenylate cyclase activator activity / protein serine/threonine kinase activator activity / VEGFR2 mediated vascular permeability / regulation of cytokinesis / VEGFR2 mediated cell proliferation / positive regulation of receptor signaling pathway via JAK-STAT / spindle microtubule
Similarity search - Function
Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / Major intrinsic protein / Major intrinsic protein / Aquaporin-like / : / EF-hand domain pair / EF-hand, calcium binding motif / EF-Hand 1, calcium-binding site ...Aquaporin transporter / Major intrinsic protein, conserved site / MIP family signature. / 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 / Lens fiber major intrinsic protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Ovis aries (sheep)
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 25 Å
AuthorsReichow, S.L. / Clemens, D.M. / Freites, J.A. / Nemeth-Cahalan, K.L. / Heyden, M. / Tobias, D.J. / Hall, J.E. / 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 31, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 31, 2013Provider: repository / Type: Initial release
Revision 1.1Aug 14, 2013Group: Database references
Revision 1.2Sep 18, 2013Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Feb 21, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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  • Deposited structure unit
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  • EMDB-5679
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Lens fiber major intrinsic protein
B: Lens fiber major intrinsic protein
C: Lens fiber major intrinsic protein
D: Lens fiber major intrinsic protein
E: Calmodulin
F: Calmodulin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)147,00514
Polymers146,6856
Non-polymers3218
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Lens fiber major intrinsic protein / Aquaporin-0


Mass: 28244.865 Da / Num. of mol.: 4 / Fragment: SEE REMARK 999 / Source method: isolated from a natural source / Source: (natural) Ovis aries (sheep) / Tissue: eye lens / References: UniProt: Q6J8I9*PLUS
#2: Protein Calmodulin / CaM


Mass: 16852.545 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human)
Gene: CALM1, CALM, CAM, CAM1, CALM2, CAM2, CAMB, CALM3, CALML2, CAM3, CAMC, CAMIII
Plasmid: pET / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: UniProt: P62158, UniProt: P0DP23*PLUS
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca
Sequence detailsCHAINS A, B, C, AND D (AQUAPORIN) ARE FROM OVIS ARIES, BUT THE MODELED SEQUENCE IS FROM BOS TAURUS ...CHAINS A, B, C, AND D (AQUAPORIN) ARE FROM OVIS ARIES, BUT THE MODELED SEQUENCE IS FROM BOS TAURUS (UNP P06624).

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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

Component
IDNameTypeDetails (eV)Parent-ID
1Aquaporin-0 bound to CalmodulinCOMPLEXOne tetramer of Aquaporin-0 bound to 2 molecules of Calmodulin0
2Aquaporin-01
3Calmodulin1
Molecular weightValue: 0.13 MDa / Experimental value: YES
Buffer solutionName: 25mM HEPES, pH 7.4, 5mM CaCl2, 0.3% decylmaltoside / pH: 7.4 / Details: 25mM HEPES, pH 7.4, 5mM CaCl2, 0.3% decylmaltoside
SpecimenConc.: 0.02 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
Details: 25mM HEPES, 5mM CaCl2, 0.3% decylmaltoside (Stain Details 0.75% Uranyl Formate)
EM stainingType: NEGATIVE / Material: Uranyl Formate
Specimen supportDetails: 400 mesh carbon coated grid (Ted Pella)

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 12 / Date: Feb 25, 2010
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM / Electron beam tilt params: 0
Electron lensMode: BRIGHT FIELD / Nominal magnification: 52000 X / Calibrated magnification: 52000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2 mm
Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
Camera length: 0 mm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: FEI Single-Tilt / Tilt angle max: 50 ° / Tilt angle min: 0 °
Image recordingElectron dose: 15 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 200
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2FREALIGN3D reconstruction
3SPIDER3D reconstruction
CTF correctionDetails: CTF-TILT, each micrograph
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionMethod: Random Conical Tilt / Resolution: 25 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 11720 / Nominal pixel size: 3.98 Å / Actual pixel size: 3.98 Å / Magnification calibration: AQP0 cyrstal
Details: Final Map with C2 Symmetry and Filtered to 25 Angstrom (Single particle details: Particles were selected from a tilted pair dataset at 0 and 50 degree tilt using SPIDER. An initial ...Details: Final Map with C2 Symmetry and Filtered to 25 Angstrom (Single particle details: Particles 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.) (Single particle--Applied symmetry: C2)
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: cross-correlation
Details: REFINEMENT PROTOCOL--rigid body DETAILS--A complete model of AQP0-CaM was built by fitting 2B6P and 1NWD into the EM map in Chimera. Loops connecting the two structures were built using COOT ...Details: REFINEMENT PROTOCOL--rigid body DETAILS--A complete model of AQP0-CaM was built by fitting 2B6P and 1NWD into the EM map in Chimera. Loops connecting the two structures were built using COOT and the final model was energy minimized to remove steric clashes. The geometries of the modeled loops (AQP0 residues 222-227) were not refined due to lack of resolution in the experimental map.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
12B6P

2b6p

A2B6P1
21NWD

1nwd

1NWD2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms9330 0 8 0 9338

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