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- PDB-3ek4: Calcium-saturated GCaMP2 Monomer -

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Basic information

Entry
Database: PDB / ID: 3ek4
TitleCalcium-saturated GCaMP2 Monomer
ComponentsMyosin light chain kinase, Green fluorescent protein, Calmodulin chimeraMyosin light-chain kinase
KeywordsFLUORESCENT PROTEIN / GECI / GCaMP2 / cpGFP / calmodulin / M13 peptide / Chromophore / Luminescence / Photoprotein / Methylation / Phosphoprotein / SIGNALING PROTEIN
Function / homology
Function and homology information


establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / establishment of protein localization to membrane / regulation of synaptic vesicle endocytosis / regulation of synaptic vesicle exocytosis / organelle localization by membrane tethering / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / nitric-oxide synthase binding ...establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / establishment of protein localization to membrane / regulation of synaptic vesicle endocytosis / regulation of synaptic vesicle exocytosis / organelle localization by membrane tethering / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / nitric-oxide synthase binding / protein phosphatase activator activity / calcium channel regulator activity / adenylate cyclase binding / catalytic complex / detection of calcium ion / negative regulation of ryanodine-sensitive calcium-release channel activity / regulation of cardiac muscle contraction / calcium channel inhibitor activity / cellular response to interferon-beta / voltage-gated potassium channel complex / phosphatidylinositol 3-kinase binding / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / positive regulation of protein dephosphorylation / titin binding / regulation of ryanodine-sensitive calcium-release channel activity / sperm midpiece / response to amphetamine / calcium channel complex / adenylate cyclase activator activity / regulation of heart rate / nitric-oxide synthase regulator activity / protein serine/threonine kinase activator activity / bioluminescence / sarcomere / regulation of cytokinesis / generation of precursor metabolites and energy / calcium-mediated signaling / spindle microtubule / positive regulation of receptor signaling pathway via JAK-STAT / spindle pole / cellular response to type II interferon / response to calcium ion / calcium-dependent protein binding / G2/M transition of mitotic cell cycle / myelin sheath / growth cone / transmembrane transporter binding / protein domain specific binding / centrosome / calcium ion binding / protein kinase binding / protein-containing complex / mitochondrion / nucleoplasm / cytosol / cytoplasm
Similarity search - Function
Green Fluorescent Protein / Green fluorescent protein / EF-hand / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Recoverin; domain 1 / EF-hand domain pair / EF-hand, calcium binding motif ...Green Fluorescent Protein / Green fluorescent protein / EF-hand / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Recoverin; domain 1 / 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 / Beta Barrel / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
Calmodulin-1 / Green fluorescent protein
Similarity search - Component
Biological speciesartificial gene (others)
Aequorea victoria (jellyfish)
Rattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.65 Å
AuthorsAkerboom, J. / Velez Rivera, J.D. / Looger, L.L. / Schreiter, E.R.
Citation
Journal: J.Biol.Chem. / Year: 2009
Title: Crystal Structures of the GCaMP Calcium Sensor Reveal the Mechanism of Fluorescence Signal Change and Aid Rational Design
Authors: Akerboom, J. / Rivera, J.D. / Guilbe, M.M. / Malave, E.C. / Hernandez, H.H. / Tian, L. / Hires, S.A. / Marvin, J.S. / Looger, L.L. / Schreiter, E.R.
#1: Journal: Acta Crystallogr.,Sect.F / Year: 2008
Title: Crystallization and preliminary X-ray characterization of the genetically encoded fluorescent calcium indicator protein GCaMP2
Authors: Rodriguez Guilbe, M.M. / Alfaro Malave, E.C. / Akerboom, J. / Marvin, J.S. / Looger, L.L. / Schreiter, E.R.
History
DepositionSep 18, 2008Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2008Provider: repository / Type: Initial release
Revision 1.1Jul 13, 2011Group: Version format compliance
Revision 1.2Oct 31, 2012Group: Database references
Revision 1.3Oct 16, 2013Group: Structure summary
Revision 1.4Jun 28, 2017Group: Database references / Source and taxonomy
Category: entity_src_gen / struct_ref ...entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _struct_ref.db_code / _struct_ref.pdbx_db_accession ..._struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.pdbx_db_accession
Revision 1.5Oct 25, 2017Group: Refinement description / Category: software
Revision 1.6Aug 30, 2023Group: 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 / 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 / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_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.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_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.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.7Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Myosin light chain kinase, Green fluorescent protein, Calmodulin chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,8254
Polymers50,7051
Non-polymers1203
Water52229
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)60.498, 117.260, 68.801
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein Myosin light chain kinase, Green fluorescent protein, Calmodulin chimera / Myosin light-chain kinase


Mass: 50704.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) artificial gene (others), (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Rattus norvegicus (Norway rat)
Plasmid: pRSETA / Gene: GFP, Calm1, Calm, Cam, Cam1, CaMI / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P42212, UniProt: P0DP29
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 29 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsRESIDUE SER65 HAS BEEN MUTATED TO GLY. RESIDUES GLY65, TYR66, AND GLY67 CONSTITUTE THE CHROMOPHORE ...RESIDUE SER65 HAS BEEN MUTATED TO GLY. RESIDUES GLY65, TYR66, AND GLY67 CONSTITUTE THE CHROMOPHORE CRO. THE AUTHOR STATES THAT ANY DIFFERENCES WITH DATABASE SEQUENCE LIKELY REPRESENT MUTATIONS INTRODUCED DURING VARIOUS ENGINEERING STEPS.

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.41 Å3/Da / Density % sol: 48.89 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8
Details: 0.1 M Magnesium formate dihydrate, 15% w/v Polyethylene glycol 3,350, pH 8, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.2.2 / Wavelength: 1 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Jun 26, 2008
RadiationMonochromator: Double crystal, Si(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 2.65→50 Å / Num. obs: 14650 / % possible obs: 98.5 % / Observed criterion σ(I): 2 / Redundancy: 3.1 % / Rmerge(I) obs: 0.076
Reflection shellResolution: 2.65→2.74 Å / Redundancy: 3 % / Rmerge(I) obs: 0.279 / Mean I/σ(I) obs: 4.2 / % possible all: 92.1

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
DENZOdata reduction
SCALEPACKdata scaling
PHASERphasing
REFMAC5.2.0019refinement
PDB_EXTRACT3.006data extraction
BOSdata collection
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 3EKH

3ekh


Resolution: 2.65→45.45 Å / Cor.coef. Fo:Fc: 0.918 / Cor.coef. Fo:Fc free: 0.881 / Occupancy max: 1 / Occupancy min: 1 / SU B: 10.669 / SU ML: 0.231 / Cross valid method: THROUGHOUT / ESU R: 0.611 / ESU R Free: 0.339 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.2795 736 5 %RANDOM
Rwork0.22229 ---
obs0.22511 13914 98.27 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 43.902 Å2
Baniso -1Baniso -2Baniso -3
1--0.69 Å20 Å20 Å2
2---2.56 Å20 Å2
3---3.25 Å2
Refinement stepCycle: LAST / Resolution: 2.65→45.45 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2767 0 3 29 2799
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.010.0222819
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.3361.973800
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.3175344
X-RAY DIFFRACTIONr_dihedral_angle_2_deg35.71625.217138
X-RAY DIFFRACTIONr_dihedral_angle_3_deg18.55315504
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.0161513
X-RAY DIFFRACTIONr_chiral_restr0.0750.2415
X-RAY DIFFRACTIONr_gen_planes_refined0.0040.022138
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined0.2040.21129
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined0.3020.21874
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1350.2113
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined0.1320.29
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1450.222
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.130.22
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.6091.51759
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it1.12222770
X-RAY DIFFRACTIONr_scbond_it1.52631173
X-RAY DIFFRACTIONr_scangle_it2.5694.51030
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.65→2.714 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.316 40 -
Rwork0.252 875 -
obs--86.57 %

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