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- PDB-9k8x: Crystal structure of the calcium indicator GCaMP6s-BrUS-145 in ca... -

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Basic information

Entry
Database: PDB / ID: 9k8x
TitleCrystal structure of the calcium indicator GCaMP6s-BrUS-145 in calcium-bounded state
ComponentsCalcium indicator GCaMP6s-BrUS-145,Calmodulin-1
KeywordsFLUORESCENT PROTEIN / Genetically encoded calcium indicator / Calcium bounded state / Calcium-sensitive fluorescence lifetime / calcium saturated
Function / homology
Function and homology information


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 ...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 / 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 calcium ion export across plasma membrane / organelle localization by membrane tethering / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / presynaptic endocytosis / regulation of cardiac muscle cell action potential / positive regulation of ryanodine-sensitive calcium-release channel activity / Synthesis of IP3 and IP4 in the cytosol / regulation of cell communication by electrical coupling involved in cardiac conduction / Phase 0 - rapid depolarisation / Negative regulation of NMDA receptor-mediated neuronal transmission / negative regulation of ryanodine-sensitive calcium-release channel activity / Unblocking of NMDA receptors, glutamate binding and activation / RHO GTPases activate PAKs / calcineurin-mediated signaling / Ion transport by P-type ATPases / Uptake and function of anthrax toxins / Long-term potentiation / Regulation of MECP2 expression and activity / Calcineurin activates NFAT / protein phosphatase activator activity / regulation of ryanodine-sensitive calcium-release channel activity / DARPP-32 events / Smooth Muscle Contraction / catalytic complex / detection of calcium ion / regulation of cardiac muscle contraction / RHO GTPases activate IQGAPs / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / presynaptic cytosol / calcium channel inhibitor activity / cellular response to interferon-beta / Protein methylation / Activation of AMPK downstream of NMDARs / Ion homeostasis / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / eNOS activation / regulation of calcium-mediated signaling / Tetrahydrobiopterin (BH4) synthesis, recycling, salvage and regulation / titin binding / voltage-gated potassium channel complex / sperm midpiece / substantia nigra development / calcium channel complex / calyx of Held / FCERI mediated Ca+2 mobilization / Ras activation upon Ca2+ influx through NMDA receptor / FCGR3A-mediated IL10 synthesis / adenylate cyclase activator activity / regulation of heart rate / Antigen activates B Cell Receptor (BCR) leading to generation of second messengers / protein serine/threonine kinase activator activity / VEGFR2 mediated cell proliferation / sarcomere / regulation of cytokinesis / VEGFR2 mediated vascular permeability / Translocation of SLC2A4 (GLUT4) to the plasma membrane / positive regulation of receptor signaling pathway via JAK-STAT / spindle microtubule / RAF activation / Transcriptional activation of mitochondrial biogenesis / Stimuli-sensing channels / cellular response to type II interferon / long-term synaptic potentiation / response to calcium ion / RAS processing / spindle pole / Signaling by RAF1 mutants / Signaling by moderate kinase activity BRAF mutants / Paradoxical activation of RAF signaling by kinase inactive BRAF / Signaling downstream of RAS mutants / calcium-dependent protein binding / G2/M transition of mitotic cell cycle / Signaling by BRAF and RAF1 fusions / Inactivation, recovery and regulation of the phototransduction cascade / Platelet degranulation / myelin sheath / Ca2+ pathway / RAF/MAP kinase cascade / High laminar flow shear stress activates signaling by PIEZO1 and PECAM1:CDH5:KDR in endothelial cells / vesicle / transmembrane transporter binding / Extra-nuclear estrogen signaling / G protein-coupled receptor signaling pathway / centrosome
Similarity search - Function
: / 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
Biological speciessynthetic construct (others)
Homo sapiens (human)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.05 Å
AuthorsVarfolomeeva, L.A. / Simonyan, T.R. / Mamontova, A.V. / Popov, V.O. / Bogdanov, A.M. / Boyko, K.M.
Funding support Russian Federation, 1items
OrganizationGrant numberCountry
Ministry of Science and Higher Education of the Russian Federation075-15-2021-1354 Russian Federation
CitationJournal: Int J Mol Sci / Year: 2024
Title: Calcium Indicators with Fluorescence Lifetime-Based Signal Readout: A Structure-Function Study.
Authors: Simonyan, T.R. / Varfolomeeva, L.A. / Mamontova, A.V. / Kotlobay, A.A. / Gorokhovatsky, A.Y. / Bogdanov, A.M. / Boyko, K.M.
History
DepositionOct 24, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 11, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 1, 2025Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Calcium indicator GCaMP6s-BrUS-145,Calmodulin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)52,45712
Polymers51,9011
Non-polymers55611
Water5,152286
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area90 Å2
ΔGint-13 kcal/mol
Surface area18200 Å2
MethodPISA
Unit cell
Length a, b, c (Å)118.293, 118.293, 98.703
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number92
Space group name H-MP41212

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Components

#1: Protein Calcium indicator GCaMP6s-BrUS-145,Calmodulin-1


Mass: 51900.863 Da / Num. of mol.: 1 / Mutation: A313P/N372D/D390Y/T391R/S393T/R402G
Source method: isolated from a genetically manipulated source
Details: Residues 1 to 46 (LAT), expression tag Residues 47 to 49 (MVD), linker Residues 50 to 70 (LSS), fragment from the protein P11799 Residues 71 to 72 (LE), linker Residues 73 to 162 (LYK), ...Details: Residues 1 to 46 (LAT), expression tag Residues 47 to 49 (MVD), linker Residues 50 to 70 (LSS), fragment from the protein P11799 Residues 71 to 72 (LE), linker Residues 73 to 162 (LYK), synthetic construct eGFP AFA52652.1 Residues 163 to 168 (GGS), linker Residues 169 to 311 (EYN), synthetic construct eGFP AFA52652.1 Residue 312 (L), linker Residues 313 to 460 (TAK), the protein P0DP23
Source: (gene. exp.) synthetic construct (others), (gene. exp.) Homo sapiens (human)
Cell (production host): XL1-Blue / Production host: Escherichia coli (E. coli) / References: UniProt: P0DP23
#2: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C2H6O2
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: Na
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 286 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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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: 3.33 Å3/Da / Density % sol: 63.01 %
Crystal growTemperature: 288 K / Method: vapor diffusion, sitting drop / Details: 0.2M Sodium thiocyanate, pH 6.9, 20% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SEALED TUBE / Type: RIGAKU PhotonJet-S / Wavelength: 1.542 Å
DetectorType: RIGAKU HyPix-6000HE / Detector: PIXEL / Date: Nov 16, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.542 Å / Relative weight: 1
ReflectionResolution: 2.05→23.01 Å / Num. obs: 44474 / % possible obs: 99.9 % / Redundancy: 24.8 % / CC1/2: 0.999 / Rmerge(I) obs: 0.184 / Rpim(I) all: 0.038 / Rrim(I) all: 0.188 / Χ2: 0.98 / Net I/σ(I): 17.2 / Num. measured all: 1103348
Reflection shellResolution: 2.05→2.11 Å / % possible obs: 100 % / Redundancy: 17.4 % / Rmerge(I) obs: 1.736 / Num. measured all: 59368 / Num. unique obs: 3411 / CC1/2: 0.83 / Rpim(I) all: 0.425 / Rrim(I) all: 1.788 / Χ2: 1.02 / Net I/σ(I) obs: 1.9

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Processing

Software
NameVersionClassification
REFMAC5.8.0430refinement
Aimlessdata scaling
CrysalisProdata reduction
MOLREPphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.05→23.01 Å / Cor.coef. Fo:Fc: 0.95 / Cor.coef. Fo:Fc free: 0.925 / SU B: 8.173 / SU ML: 0.114 / Cross valid method: THROUGHOUT / ESU R: 0.157 / ESU R Free: 0.152 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.25065 2167 5 %RANDOM
Rwork0.21057 ---
obs0.21264 41351 97.87 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 34.733 Å2
Baniso -1Baniso -2Baniso -3
1-0.29 Å2-0 Å2-0 Å2
2--0.29 Å2-0 Å2
3----0.57 Å2
Refinement stepCycle: 1 / Resolution: 2.05→23.01 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3089 0 48 286 3423
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0160.0123244
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg2.6031.8384328
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg5.4525397
X-RAY DIFFRACTIONr_dihedral_angle_2_deg5.628522
X-RAY DIFFRACTIONr_dihedral_angle_3_deg10.89310549
X-RAY DIFFRACTIONr_dihedral_angle_4_deg
X-RAY DIFFRACTIONr_chiral_restr0.1740.2472
X-RAY DIFFRACTIONr_gen_planes_refined0.0140.022443
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it3.5722.6071601
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it4.8964.6681988
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it5.2432.811643
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined8.53527.024880
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 2.05→2.103 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.377 164 -
Rwork0.282 3030 -
obs--99.19 %
Refinement TLS params.Method: refined / Origin x: -3.913 Å / Origin y: 39.38 Å / Origin z: 1.679 Å
111213212223313233
T0.0134 Å2-0.0013 Å2-0.0018 Å2-0.0082 Å20.0105 Å2--0.0152 Å2
L0.8037 °2-0.3201 °20.243 °2-0.4796 °2-0.0831 °2--1.0654 °2
S0.0152 Å °-0.0495 Å °-0.0632 Å °0.0431 Å °0.0145 Å °0.0341 Å °0.0815 Å °-0.0001 Å °-0.0297 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A47 - 460
2X-RAY DIFFRACTION1A501 - 506

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