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- PDB-3sg6: Crystal Structure of Dimeric GCaMP2-LIA(linker 1) -

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Basic information

Entry
Database: PDB / ID: 3sg6
TitleCrystal Structure of Dimeric GCaMP2-LIA(linker 1)
ComponentsMyosin light chain kinase, Green fluorescent protein, Calmodulin-1 chimeraMyosin light-chain kinase
KeywordsFLUORESCENT PROTEIN / calcium sensor
Function / homology
Function and homology information


tonic smooth muscle contraction / myosin-light-chain kinase / myosin light chain kinase activity / 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 ...tonic smooth muscle contraction / myosin-light-chain kinase / myosin light chain kinase activity / 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 / cleavage furrow / calcium channel regulator activity / adenylate cyclase binding / catalytic complex / detection of calcium ion / negative regulation of ryanodine-sensitive calcium-release channel activity / cellular response to interferon-beta / regulation of cardiac muscle contraction / calcium channel inhibitor activity / voltage-gated potassium channel complex / phosphatidylinositol 3-kinase binding / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / stress fiber / response to amphetamine / positive regulation of protein dephosphorylation / titin binding / regulation of ryanodine-sensitive calcium-release channel activity / sperm midpiece / calcium channel complex / adenylate cyclase activator activity / protein serine/threonine kinase activator activity / regulation of heart rate / nitric-oxide synthase regulator activity / sarcomere / bioluminescence / regulation of cytokinesis / generation of precursor metabolites and energy / calcium-mediated signaling / spindle microtubule / positive regulation of receptor signaling pathway via JAK-STAT / cellular response to type II interferon / spindle pole / response to calcium ion / calcium-dependent protein binding / G2/M transition of mitotic cell cycle / lamellipodium / myelin sheath / kinase activity / 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 / cytosol / cytoplasm
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 / Green fluorescent protein, GFP / EF-hand / Green fluorescent protein-related / Green fluorescent protein ...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 / Green fluorescent protein, GFP / EF-hand / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Recoverin; domain 1 / 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 / Myosin light chain kinase
Similarity search - Component
Biological speciesGallus gallus (chicken)
Aequorea victoria (jellyfish)
Rattus norvegicus (Norway rat)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.7 Å
AuthorsSchreiter, E.R. / Akerboom, J. / Looger, L.L.
CitationJournal: J.Neurosci. / Year: 2012
Title: Optimization of a GCaMP calcium indicator for neural activity imaging.
Authors: Akerboom, J. / Chen, T.W. / Wardill, T.J. / Tian, L. / Marvin, J.S. / Mutlu, S. / Calderon, N.C. / Esposti, F. / Borghuis, B.G. / Sun, X.R. / Gordus, A. / Orger, M.B. / Portugues, R. / ...Authors: Akerboom, J. / Chen, T.W. / Wardill, T.J. / Tian, L. / Marvin, J.S. / Mutlu, S. / Calderon, N.C. / Esposti, F. / Borghuis, B.G. / Sun, X.R. / Gordus, A. / Orger, M.B. / Portugues, R. / Engert, F. / Macklin, J.J. / Filosa, A. / Aggarwal, A. / Kerr, R.A. / Takagi, R. / Kracun, S. / Shigetomi, E. / Khakh, B.S. / Baier, H. / Lagnado, L. / Wang, S.S. / Bargmann, C.I. / Kimmel, B.E. / Jayaraman, V. / Svoboda, K. / Kim, D.S. / Schreiter, E.R. / Looger, L.L.
History
DepositionJun 14, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 20, 2012Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2014Group: Database references
Revision 1.2Jun 28, 2017Group: Database references / Source and taxonomy / Structure summary
Category: entity / entity_name_com ...entity / entity_name_com / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _entity.pdbx_description / _struct_ref.db_code ..._entity.pdbx_description / _struct_ref.db_code / _struct_ref.pdbx_db_accession / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq_dif.align_id / _struct_ref_seq_dif.details / _struct_ref_seq_dif.mon_id / _struct_ref_seq_dif.pdbx_auth_seq_num / _struct_ref_seq_dif.pdbx_seq_db_accession_code / _struct_ref_seq_dif.seq_num
Revision 1.3Sep 13, 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_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_seq_id / _pdbx_struct_conn_angle.ptnr2_label_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.pdbx_leaving_atom_flag / _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_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_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.4Dec 6, 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-1 chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,9205
Polymers50,7601
Non-polymers1604
Water4,900272
1
A: Myosin light chain kinase, Green fluorescent protein, Calmodulin-1 chimera
hetero molecules

A: Myosin light chain kinase, Green fluorescent protein, Calmodulin-1 chimera
hetero molecules


Theoretical massNumber of molelcules
Total (without water)101,84010
Polymers101,5192
Non-polymers3218
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area7490 Å2
ΔGint-163 kcal/mol
Surface area35000 Å2
MethodPISA
Unit cell
Length a, b, c (Å)128.976, 47.491, 68.673
Angle α, β, γ (deg.)90.00, 99.72, 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-1 chimera / Myosin light-chain kinase


Mass: 50759.707 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999 / Mutation: LIA(linker 1)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken), (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Rattus norvegicus (Norway rat)
Gene: GFP, Calm1, Calm, Cam, Cam1, CaMI / Production host: Escherichia coli (E. coli)
References: UniProt: Q6LDG3, 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 / Water


Mass: 18.015 Da / Num. of mol.: 272 / Source method: isolated from a natural source / Formula: H2O
Nonpolymer detailsTHIS TRIPLE CHIMERA COMPRISES (FROM THE N-TERMINUS) AN ENGINEERED EXPRESSION TAG, RESIDUES 37-55 OF ...THIS TRIPLE CHIMERA COMPRISES (FROM THE N-TERMINUS) AN ENGINEERED EXPRESSION TAG, RESIDUES 37-55 OF UNP Q6LDG3, AN ENGINEERED LIA LINKER, RESIDUES 149-239 OF UNP P42212, AN ENGINEERED GGTGGSMV LINKER, RESIDUES 2-144 OF UNP P42212, AN ENGINEERED TR LINKER, AND RESIDUES 3-149 OF UNP P62161. UNP P42212 RESIDUES S65, Y66, AND G67 ARE THE CHROMOPHORE CRO.

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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: 2.04 Å3/Da / Density % sol: 39.76 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 0.2 M lithium sulfate, 0.1 M Tris, pH 8.5, 30% PEG4000, VAPOR DIFFUSION, SITTING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU RUH3R / Wavelength: 1.5418
DetectorType: RIGAKU SATURN 92 / Detector: CCD / Date: Jul 2, 2007 / Details: mirrors
RadiationMonochromator: osmic mirrors / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.7→28 Å / Num. all: 43065 / Num. obs: 40438 / % possible obs: 93.9 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 7.2 % / Rsym value: 0.07 / Net I/σ(I): 15.9
Reflection shellResolution: 1.7→1.76 Å / Redundancy: 7.1 % / Mean I/σ(I) obs: 1.6 / Rsym value: 0.468 / % possible all: 73.9

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Processing

Software
NameVersionClassification
CrystalCleardata collection
PHASERphasing
REFMAC5.6.0111refinement
d*TREKdata reduction
d*TREKdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3EK7
Resolution: 1.7→27.74 Å / Cor.coef. Fo:Fc: 0.957 / Cor.coef. Fo:Fc free: 0.935 / SU B: 4.976 / SU ML: 0.084 / Cross valid method: THROUGHOUT / ESU R: 0.127 / ESU R Free: 0.127 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT U VALUES : RESIDUAL ONLY
RfactorNum. reflection% reflectionSelection details
Rfree0.24942 2147 5 %RANDOM
Rwork0.20339 ---
all0.20573 43065 --
obs0.20573 40438 93.84 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 31.106 Å2
Baniso -1Baniso -2Baniso -3
1-0.1 Å20 Å20.05 Å2
2---0.49 Å20 Å2
3---0.4 Å2
Refinement stepCycle: LAST / Resolution: 1.7→27.74 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3057 0 4 272 3333
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0250.0223142
X-RAY DIFFRACTIONr_bond_other_d0.0010.022136
X-RAY DIFFRACTIONr_angle_refined_deg2.0181.9674242
X-RAY DIFFRACTIONr_angle_other_deg1.08135223
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.3285388
X-RAY DIFFRACTIONr_dihedral_angle_2_deg33.84125.313160
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.20215567
X-RAY DIFFRACTIONr_dihedral_angle_4_deg22.3651516
X-RAY DIFFRACTIONr_chiral_restr0.1270.2462
X-RAY DIFFRACTIONr_gen_planes_refined0.010.023519
X-RAY DIFFRACTIONr_gen_planes_other0.0030.02621
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_it
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it
X-RAY DIFFRACTIONr_scbond_it
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.7→1.744 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.359 138 -
Rwork0.348 2228 -
obs--72.69 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL112)L122)L132)L222)L232)L332)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T112)T122)T132)T222)T232)T332)Origin x (Å)Origin y (Å)Origin z (Å)
16.8516-1.6848-5.76926.07963.20995.73490.2994-0.43690.3829-0.34890.2126-0.3288-0.3470.5693-0.5120.14120.1054-0.00720.2971-0.12310.0945-16.14318.71610.488
21.0262-1.2263-0.13355.77580.91970.8534-0.0422-0.171-0.09560.111-0.01710.32450.02970.02070.05930.0873-0.0101-0.00350.08850.01710.0766-16.9541.809-14.777
31.2508-0.06220.19211.63950.33781.31440.057-0.0993-0.0674-0.0238-0.01230.0072-0.02790.0516-0.04470.0928-0.01770.01090.08930.0250.065-15.0362.914-17.652
41.0077-0.05260.10670.9045-0.26290.9140.0390.00790.0268-0.0431-0.036-0.0198-0.03720.0856-0.0030.0857-0.0171-0.00080.0827-0.00090.0692-12.3549.969-25.78
53.49210.92080.932.36171.67632.0993-0.03270.16120.3625-0.0846-0.27320.0833-0.0104-0.11260.30590.0689-0.0943-0.01580.24830.07340.101613.56510.098-13.345
62.2386-0.58351.81821.6218-1.78824.9748-0.06340.03720.08870.13590.12830.090.1826-0.4472-0.06480.0713-0.0882-0.00040.28710.02620.01328.5556.553-4.779
73.80210.2721-0.36474.3005-0.09562.49250.149-0.2691-0.36440.5177-0.171-0.38790.1779-0.18550.02190.1337-0.1149-0.07850.13540.06680.073326.47319.801-7.385
81.69541.83490.5242.24150.86142.63840.1423-0.04410.20110.0736-0.00880.20320.0145-0.3597-0.13340.0683-0.0296-0.00210.12410.01390.054626.728.305-13.932
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A40 - 60
2X-RAY DIFFRACTION2A61 - 85
3X-RAY DIFFRACTION3A86 - 119
4X-RAY DIFFRACTION4A120 - 303
5X-RAY DIFFRACTION5A307 - 345
6X-RAY DIFFRACTION6A346 - 378
7X-RAY DIFFRACTION7A385 - 419
8X-RAY DIFFRACTION8A420 - 450

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