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- PDB-3wld: Crystal structure of monomeric GCaMP6m -

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Basic information

Entry
Database: PDB / ID: 3wld
TitleCrystal structure of monomeric GCaMP6m
ComponentsMyosin light chain kinase, Green fluorescent protein, Calmodulin
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 / negative regulation of ryanodine-sensitive calcium-release channel activity / organelle localization by membrane tethering / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking ...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 / negative regulation of ryanodine-sensitive calcium-release channel activity / organelle localization by membrane tethering / mitochondrion-endoplasmic reticulum membrane tethering / autophagosome membrane docking / negative regulation of calcium ion export across plasma membrane / regulation of cardiac muscle cell action potential / presynaptic endocytosis / nitric-oxide synthase binding / regulation of synaptic vesicle exocytosis / calcineurin-mediated signaling / cleavage furrow / adenylate cyclase binding / regulation of ryanodine-sensitive calcium-release channel activity / protein phosphatase activator activity / catalytic complex / detection of calcium ion / regulation of synaptic vesicle endocytosis / regulation of cardiac muscle contraction / postsynaptic cytosol / cellular response to interferon-beta / phosphatidylinositol 3-kinase binding / calcium channel inhibitor activity / presynaptic cytosol / stress fiber / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / titin binding / sperm midpiece / regulation of calcium-mediated signaling / voltage-gated potassium channel complex / calcium channel complex / regulation of heart rate / calyx of Held / bioluminescence / response to amphetamine / adenylate cyclase activator activity / sarcomere / nitric-oxide synthase regulator activity / protein serine/threonine kinase activator activity / regulation of cytokinesis / generation of precursor metabolites and energy / spindle microtubule / calcium channel regulator activity / positive regulation of receptor signaling pathway via JAK-STAT / response to calcium ion / cellular response to type II interferon / G2/M transition of mitotic cell cycle / Schaffer collateral - CA1 synapse / spindle pole / calcium-dependent protein binding / kinase activity / myelin sheath / lamellipodium / growth cone / vesicle / transmembrane transporter binding / calmodulin binding / protein domain specific binding / calcium ion binding / centrosome / 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 / : / Green fluorescent protein, GFP / 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 / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Fibronectin type III domain / Fibronectin type 3 domain / Immunoglobulin subtype 2 / Immunoglobulin C-2 Type / EF-hand domain pair / 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 / Immunoglobulin-like domain superfamily / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / 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 / Mainly Beta
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: 2.7 Å
AuthorsDing, J. / Luo, A.F. / Hu, L.Y. / Wang, D.C. / Shao, F.
CitationJournal: Sci China Life Sci / Year: 2014
Title: Structural basis of the ultrasensitive calcium indicator GCaMP6.
Authors: Ding, J. / Luo, A.F. / Hu, L. / Wang, D. / Shao, F.
History
DepositionNov 8, 2013Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 22, 2014Provider: repository / Type: Initial release
Revision 1.1Jun 21, 2017Group: Database references / Source and taxonomy / Structure summary
Category: entity / entity_src_gen ...entity / entity_src_gen / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _entity.details / _struct_ref.db_code ..._entity.details / _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.pdbx_seq_db_accession_code
Revision 1.2Aug 24, 2022Group: Database references / Derived calculations
Category: citation / database_2 ...citation / database_2 / pdbx_struct_conn_angle / struct_conn / struct_site
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _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 1.3Nov 8, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
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
Revision 1.5Oct 9, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)50,5585
Polymers50,3981
Non-polymers1604
Water2,918162
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)120.880, 120.880, 97.620
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 Myosin light chain kinase, Green fluorescent protein, Calmodulin / CaM


Mass: 50398.176 Da / Num. of mol.: 1 / Fragment: UNP RESIDUES 37-55, 149-238, 2-144, 3-149
Mutation: M153K, V163A, S175G, D180Y, T203V, A206K, H231L, F64L, V93I, N61D, D79Y, M77G, K78S, T80R, S82T, R91G
Source method: isolated from a genetically manipulated source
Details: THIS TRIPLE CHIMERA COMPRISES (FROM THE N-TERMINUS) AN ENGINEERED EXPRESSION TAG, RESIDUES 37-55 OF UNP Q6LDG3, AN ENGINEERED LE LINKER, RESIDUES 149-239 OF UNP P42212, AN ENGINEERED ...Details: THIS TRIPLE CHIMERA COMPRISES (FROM THE N-TERMINUS) AN ENGINEERED EXPRESSION TAG, RESIDUES 37-55 OF UNP Q6LDG3, AN ENGINEERED LE LINKER, RESIDUES 149-239 OF UNP P42212, AN ENGINEERED GGTGGSMV LINKER, RESIDUES 2-144 OF UNP P42212, AN ENGINEERED LP LINKER, AND RESIDUES 3-149 OF UNP P0DP29. UNP P42212 RESIDUES S65, Y66, AND G67 ARE THE CHROMOPHORE CRO.
Source: (gene. exp.) Gallus gallus (chicken), (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Rattus norvegicus (Norway rat)
Plasmid: pSUMO / Gene: GFP, Calm, Cam / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)
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


Mass: 18.015 Da / Num. of mol.: 162 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY
Sequence details(1)THIS TRIPLE CHIMERA COMPRISES (FROM THE N-TERMINUS) AN ENGINEERED EXPRESSION TAG, RESIDUES 37-55 ...(1)THIS TRIPLE CHIMERA COMPRISES (FROM THE N-TERMINUS) AN ENGINEERED EXPRESSION TAG, RESIDUES 37-55 OF UNP Q6LDG3, AN ENGINEERED LE LINKER, RESIDUES 149-238 OF UNP P42212, AN ENGINEERED GGTGGSMV LINKER, RESIDUES 2-144 OF UNP P42212, AN ENGINEERED LP LINKER, AND RESIDUES 3-149 OF UNP P62161. (2)RESIDUE SER 65 HAS BEEN MUTATED TO GLY 65. RESIDUES GLY 65, TYR 66 AND GLY 67 CONSTITUTE THE CHROMOPHORE CRO 66.

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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: 3.54 Å3/Da / Density % sol: 65.23 %
Crystal growTemperature: 293 K / Method: vapor diffusion, hanging drop / pH: 7.4
Details: 0.1M HEPES, 18% w/v PEG 3350, pH 7.4, VAPOR DIFFUSION, HANGING DROP, temperature 293K

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Data collection

DiffractionMean temperature: 95 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU FR-E+ DW / Wavelength: 1.5418 Å
DetectorType: RIGAKU RAXIS IV++ / Detector: IMAGE PLATE / Date: Aug 28, 2013
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.7→60.44 Å / Num. all: 20398 / Num. obs: 20011 / % possible obs: 98.1 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 5.3 % / Biso Wilson estimate: 32.1 Å2 / Rmerge(I) obs: 0.087 / Rsym value: 0.087 / Net I/σ(I): 12.7
Reflection shellResolution: 2.7→2.85 Å / Redundancy: 4.1 % / Rmerge(I) obs: 0.377 / Mean I/σ(I) obs: 3.3 / Num. unique all: 2774 / Rsym value: 0.377 / % possible all: 95.3

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Processing

Software
NameVersionClassification
CrystalCleardata collection
PHASERphasing
PHENIX(phenix.refine: 1.8.2_1309)refinement
MOSFLMdata reduction
SCALAdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 3SG4

3sg4


Resolution: 2.7→54.059 Å / SU ML: 0.29 / σ(F): 1.34 / Phase error: 20.46 / Stereochemistry target values: ML
RfactorNum. reflection% reflectionSelection details
Rfree0.2121 1020 5.11 %RANDOM
Rwork0.17 ---
all0.1722 20439 --
obs0.1722 19975 97.73 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine analyzeLuzzati coordinate error obs: 0.29 Å
Refinement stepCycle: LAST / Resolution: 2.7→54.059 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3133 0 4 162 3299
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0063192
X-RAY DIFFRACTIONf_angle_d1.0844304
X-RAY DIFFRACTIONf_dihedral_angle_d14.8311200
X-RAY DIFFRACTIONf_chiral_restr0.066464
X-RAY DIFFRACTIONf_plane_restr0.004569
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Total num. of bins used: 7

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.7001-2.84250.30811450.231256095
2.8425-3.02050.26671350.2137258395
3.0205-3.25370.23451530.1807262497
3.2537-3.58110.21631540.1622270899
3.5811-4.09920.18031400.15052773100
4.0992-5.16390.17461650.13742772100
5.1639-54.070.21831280.1872293599

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