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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


gap junction-mediated intercellular transport / water channel activity / water transport / : / structural constituent of eye lens / gap junction / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / response to stimulus / CaM pathway ...gap junction-mediated intercellular transport / water channel activity / water transport / : / structural constituent of eye lens / gap junction / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / response to stimulus / CaM pathway / response to corticosterone / Cam-PDE 1 activation / Sodium/Calcium exchangers / lens development in camera-type eye / Calmodulin induced events / Reduction of cytosolic Ca++ levels / Activation of Ca-permeable Kainate Receptor / regulation of synaptic vesicle endocytosis / CREB1 phosphorylation through the activation of CaMKII/CaMKK/CaMKIV cascasde / Loss of phosphorylation of MECP2 at T308 / PKA activation / CREB1 phosphorylation through the activation of Adenylate Cyclase / negative regulation of high voltage-gated calcium channel activity / CaMK IV-mediated phosphorylation of CREB / Glycogen breakdown (glycogenolysis) / regulation of synaptic vesicle exocytosis / negative regulation of calcium ion export across plasma membrane / organelle localization by membrane tethering / Activation of RAC1 downstream of NMDARs / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / CLEC7A (Dectin-1) induces NFAT activation / autophagosome membrane docking / Negative regulation of NMDA receptor-mediated neuronal transmission / positive regulation of ryanodine-sensitive calcium-release channel activity / nitric-oxide synthase binding / Unblocking of NMDA receptors, glutamate binding and activation / regulation of cell communication by electrical coupling involved in cardiac conduction / negative regulation of peptidyl-threonine phosphorylation / Synthesis of IP3 and IP4 in the cytosol / Phase 0 - rapid depolarisation / protein phosphatase activator activity / RHO GTPases activate PAKs / positive regulation of cyclic-nucleotide phosphodiesterase activity / Long-term potentiation / positive regulation of phosphoprotein phosphatase activity / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / Regulation of MECP2 expression and activity / Calcineurin activates NFAT / adenylate cyclase binding / catalytic complex / detection of calcium ion / DARPP-32 events / positive regulation of cell adhesion / Smooth Muscle Contraction / negative regulation of ryanodine-sensitive calcium-release channel activity / RHO GTPases activate IQGAPs / cellular response to interferon-beta / regulation of cardiac muscle contraction / calcium channel inhibitor activity / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / positive regulation of DNA binding / Protein methylation / enzyme regulator activity / voltage-gated potassium channel complex / Activation of AMPK downstream of NMDARs / phosphatidylinositol 3-kinase binding / eNOS activation / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / response to amphetamine / regulation of calcium-mediated signaling / positive regulation of protein dephosphorylation / titin binding / regulation of ryanodine-sensitive calcium-release channel activity / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / Ion homeostasis / positive regulation of protein autophosphorylation / sperm midpiece / calcium channel complex / activation of adenylate cyclase activity / substantia nigra development / visual perception / adenylate cyclase activator activity / Ras activation upon Ca2+ influx through NMDA receptor / protein serine/threonine kinase activator activity / regulation of heart rate / nitric-oxide synthase regulator activity / sarcomere / FCERI mediated Ca+2 mobilization / FCGR3A-mediated IL10 synthesis / VEGFR2 mediated vascular permeability / positive regulation of peptidyl-threonine phosphorylation / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / regulation of cytokinesis / VEGFR2 mediated cell proliferation / positive regulation of nitric-oxide synthase activity / Translocation of SLC2A4 (GLUT4) to the plasma membrane / spindle microtubule / mitochondrial membrane
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 / EF-hand calcium-binding domain. ...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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  • Superimposition on EM map
  • EMDB-5679
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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
Water0
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 lensLens (anatomy) / 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
IDNameTypeDetailsParent-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 FIELDBright-field microscopy / 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
12B6PA2B6P1
21NWD1NWD2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms9330 0 8 0 9338

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