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- PDB-3ek7: Calcium-saturated GCaMP2 dimer -

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Basic information

Entry
Database: PDB / ID: 3ek7
TitleCalcium-saturated GCaMP2 dimer
ComponentsMyosin light chain kinase, Green fluorescent protein, Calmodulin chimera
KeywordsFLUORESCENT PROTEIN / GECI / GCaMP2 / cpEGFP / calmodulin / M13 peptide / Methylation / Phosphoprotein / SIGNALING PROTEIN
Function / homology
Function and homology information


tonic smooth muscle contraction / myosin-light-chain kinase / myosin light chain kinase activity / muscle structure development / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / establishment of protein localization to membrane / regulation of synaptic vesicle endocytosis / organelle localization by membrane tethering / regulation of synaptic vesicle exocytosis ...tonic smooth muscle contraction / myosin-light-chain kinase / myosin light chain kinase activity / muscle structure development / establishment of protein localization to mitochondrial membrane / type 3 metabotropic glutamate receptor binding / establishment of protein localization to membrane / regulation of synaptic vesicle endocytosis / organelle localization by membrane tethering / regulation of synaptic vesicle exocytosis / regulation of cardiac muscle cell action potential / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / nitric-oxide synthase binding / protein phosphatase activator activity / cleavage furrow / adenylate cyclase binding / catalytic complex / calcium channel regulator activity / detection of calcium ion / regulation of cardiac muscle contraction / negative regulation of ryanodine-sensitive calcium-release channel activity / calcium channel inhibitor activity / cellular response to interferon-beta / phosphatidylinositol 3-kinase binding / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / stress fiber / positive regulation of protein dephosphorylation / regulation of ryanodine-sensitive calcium-release channel activity / titin binding / voltage-gated potassium channel complex / sperm midpiece / calcium channel complex / response to amphetamine / adenylate cyclase activator activity / nitric-oxide synthase regulator activity / regulation of heart rate / sarcomere / protein serine/threonine kinase activator activity / bioluminescence / regulation of cytokinesis / generation of precursor metabolites and energy / positive regulation of receptor signaling pathway via JAK-STAT / spindle microtubule / cellular response to type II interferon / spindle pole / response to calcium ion / G2/M transition of mitotic cell cycle / calcium-dependent protein binding / lamellipodium / myelin sheath / growth cone / transmembrane transporter binding / calmodulin binding / protein domain specific binding / phosphorylation / centrosome / calcium ion binding / protein kinase binding / protein-containing complex / mitochondrion / nucleoplasm / ATP binding / metal ion binding / cytoplasm / cytosol
Similarity search - Function
Myosin Light Chain Kinase 1, Kinase domain / Unstructured linker between I-set domains 2 and 3 on MYLCK / Green Fluorescent Protein / Green fluorescent protein / Immunoglobulin I-set / Immunoglobulin I-set domain / EF-hand / Green fluorescent protein, GFP / Recoverin; domain 1 / Green fluorescent protein-related ...Myosin Light Chain Kinase 1, Kinase domain / Unstructured linker between I-set domains 2 and 3 on MYLCK / Green Fluorescent Protein / Green fluorescent protein / Immunoglobulin I-set / Immunoglobulin I-set domain / EF-hand / Green fluorescent protein, GFP / Recoverin; domain 1 / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Fibronectin type III domain / Fibronectin type 3 domain / EF-hand domain pair / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / Fibronectin type-III domain profile. / Fibronectin type III / Fibronectin type III superfamily / 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 / Immunoglobulin subtype / Immunoglobulin / Ig-like domain profile. / Immunoglobulin-like domain / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Immunoglobulin-like domain superfamily / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Immunoglobulin-like fold / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily / Beta Barrel / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
Calmodulin-1 / Myosin light chain kinase, smooth muscle / 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: 1.85 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: M Rodriguez Guilbe, 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 1.3Jun 21, 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 1.4Oct 25, 2017Group: Refinement description / Category: software
Revision 1.5Aug 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 / struct_conn / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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_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 1.6Nov 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)50,8655
Polymers50,7051
Non-polymers1604
Water3,567198
1
A: Myosin light chain kinase, Green fluorescent protein, Calmodulin chimera
hetero molecules

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


Theoretical massNumber of molelcules
Total (without water)101,73010
Polymers101,4092
Non-polymers3218
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area7120 Å2
ΔGint-153 kcal/mol
Surface area35270 Å2
MethodPISA
Unit cell
Length a, b, c (Å)126.131, 47.310, 68.940
Angle α, β, γ (deg.)90.00, 100.48, 90.00
Int Tables number5
Space group name H-MC121

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Components

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


Mass: 50704.586 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: calcium-sensor; protein contains circular permuted EGFP, the M13 fragment of myosin light chain kinase, rat calmodulin and a 6x His-tag from the pRSETA vector
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, UniProt: P11799*PLUS
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 198 / 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: 1.99 Å3/Da / Density % sol: 38.06 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 8.5
Details: 0.2 M lithium sulfate monohydrate, 0.1 M Tris-HCl pH 8.5, 30%(w/v) polyethylene glycol 4000, VAPOR DIFFUSION, HANGING DROP, temperature 298K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 31-ID / Wavelength: 0.9793 Å
DetectorType: MAR CCD 165 mm / Detector: CCD / Date: Jul 28, 2008
RadiationMonochromator: Diamond(1,1,1) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9793 Å / Relative weight: 1
ReflectionResolution: 1.85→67.73 Å / Num. obs: 36788 / % possible obs: 98.49 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 7.5 % / Rsym value: 0.076
Reflection shellResolution: 1.85→1.9 Å / Redundancy: 7.6 % / Mean I/σ(I) obs: 5.1 / Rsym value: 0.42 / % possible all: 97.6

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
SCALAdata scaling
PHASERphasing
REFMAC5.2.0019refinement
PDB_EXTRACT3.006data extraction
DENZOdata reduction
SCALEPACKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entries 1EMA and 1CDL

1ema

1cdl


Resolution: 1.85→20.67 Å / Cor.coef. Fo:Fc: 0.951 / Cor.coef. Fo:Fc free: 0.923 / Occupancy max: 1 / Occupancy min: 0.4 / SU B: 3.064 / SU ML: 0.095 / Cross valid method: THROUGHOUT / ESU R: 0.15 / ESU R Free: 0.146 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.24052 1714 5.1 %RANDOM
Rwork0.18871 ---
obs0.19129 32223 98.49 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 20.837 Å2
Baniso -1Baniso -2Baniso -3
1--0.09 Å20 Å2-0.57 Å2
2--0.6 Å20 Å2
3----0.73 Å2
Refinement stepCycle: LAST / Resolution: 1.85→20.67 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3105 0 4 198 3307
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0223191
X-RAY DIFFRACTIONr_bond_other_d0.0020.022172
X-RAY DIFFRACTIONr_angle_refined_deg1.7351.9684305
X-RAY DIFFRACTIONr_angle_other_deg0.98635311
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.1995393
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.39125.312160
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.39315579
X-RAY DIFFRACTIONr_dihedral_angle_4_deg17.071516
X-RAY DIFFRACTIONr_chiral_restr0.0970.2467
X-RAY DIFFRACTIONr_gen_planes_refined0.0070.023582
X-RAY DIFFRACTIONr_gen_planes_other0.0020.02628
X-RAY DIFFRACTIONr_nbd_refined0.2180.2648
X-RAY DIFFRACTIONr_nbd_other0.1990.22115
X-RAY DIFFRACTIONr_nbtor_refined0.1780.21506
X-RAY DIFFRACTIONr_nbtor_other0.1110.21719
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.150.2161
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined0.1540.212
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.1480.218
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2740.263
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.5270.218
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it1.4041.52027
X-RAY DIFFRACTIONr_mcbond_other0.3161.5808
X-RAY DIFFRACTIONr_mcangle_it1.99123134
X-RAY DIFFRACTIONr_scbond_it3.231344
X-RAY DIFFRACTIONr_scangle_it4.6994.51170
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.85→1.898 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.285 121 -
Rwork0.207 2378 -
obs--97.24 %

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