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- PDB-1huy: CRYSTAL STRUCTURE OF CITRINE, AN IMPROVED YELLOW VARIANT OF GREEN... -

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Basic information

Entry
Database: PDB / ID: 1huy
TitleCRYSTAL STRUCTURE OF CITRINE, AN IMPROVED YELLOW VARIANT OF GREEN FLUORESCENT PROTEIN
ComponentsGREEN FLUORESCENT PROTEIN
KeywordsLUMINESCENT PROTEIN / beta barrel / chromophore
Function / homology
Function and homology information


bioluminescence / generation of precursor metabolites and energy
Similarity search - Function
Green Fluorescent Protein / Green fluorescent protein / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Green fluorescent protein
Similarity search - Component
Biological speciesAequorea victoria (jellyfish)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 2.2 Å
AuthorsGriesbeck, O. / Baird, G.S. / Campbell, R.E. / Zacharias, D.A. / Tsien, R.Y.
CitationJournal: J.Biol.Chem. / Year: 2001
Title: Reducing the environmental sensitivity of yellow fluorescent protein. Mechanism and applications
Authors: Griesbeck, O. / Baird, G.S. / Campbell, R.E. / Zacharias, D.A. / Tsien, R.Y.
History
DepositionJan 4, 2001Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 4, 2001Provider: repository / Type: Initial release
Revision 1.1Apr 27, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Oct 4, 2017Group: Refinement description / Category: software
Revision 1.4Oct 27, 2021Group: Database references / Derived calculations / Category: database_2 / struct_conn / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_leaving_atom_flag / _struct_ref_seq_dif.details
Revision 1.5Aug 9, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
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
Revision 1.7Nov 20, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature
Remark 600HETEROGEN THE HET RESIDUE CRO IS FORMED FROM AN AUTOCATALYTIC POST-TRANSLATIONAL CYCLIZATION, ...HETEROGEN THE HET RESIDUE CRO IS FORMED FROM AN AUTOCATALYTIC POST-TRANSLATIONAL CYCLIZATION, DEHYDRATION, AND OXIDATION OF RESIDUES GLY 65, TYR 66, AND GLY 67, RESULTING IN A COVALENTLY LINKED GREEN CHROMOPHORE.
Remark 999SEQUENCE RESIDUES GLY65, TYR66, AND GLY67 ARE NOT PRESENT IN THE ENTRY. INSTEAD THEY ARE REPLACED ...SEQUENCE RESIDUES GLY65, TYR66, AND GLY67 ARE NOT PRESENT IN THE ENTRY. INSTEAD THEY ARE REPLACED WITH CRO66. The GFP chromophore (CRO) is formed from cyclization of the main chain of residues Gly65, Tyr66, and Gly67. A subsequent oxidation of the Tyr66 side chain yields the mature chromophore. The N-terminal addition of an Asp and a Pro residue results from Enterokinase catalyzed cleavage of a His tag that facilitated purification. Val 1A is inserted to improve expression in mammalian systems.

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: GREEN FLUORESCENT PROTEIN


Theoretical massNumber of molelcules
Total (without water)27,3201
Polymers27,3201
Non-polymers00
Water1,74797
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)52.504, 61.758, 70.684
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein GREEN FLUORESCENT PROTEIN


Mass: 27319.883 Da / Num. of mol.: 1 / Mutation: S65G, V68L, Q69M, S72A, T203Y
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Plasmid: PRSETB / Production host: Escherichia coli (E. coli) / Strain (production host): JM109(DE3) / References: UniProt: P42212
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 97 / Source method: isolated from a natural source / Formula: H2O
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: 2.1 Å3/Da / Density % sol: 41.36 %
Crystal growTemperature: 277 K / Method: vapor diffusion, hanging drop / pH: 5
Details: 50 mM NH4OAc, 50 mM NaOAc, 8% PEG 3400, pH 5.0, VAPOR DIFFUSION, HANGING DROP, temperature 277K
Crystal grow
*PLUS
Temperature: 4 ℃
Components of the solutions
*PLUS
IDConc.Common nameCrystal-IDSol-IDChemical formula
120 mg/mlprotein1drop
27 %PEG34001reservoir
350 mM1reservoirNH4OAc
450 mM1reservoirNaOAc

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Data collection

DiffractionMean temperature: 298 K
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU / Wavelength: 1.5418 Å
DetectorType: MARRESEARCH / Detector: IMAGE PLATE / Date: Nov 17, 2000 / Details: mirrors
RadiationMonochromator: GRAPHITE / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 2.2→50 Å / Num. all: 12193 / Num. obs: 12106 / % possible obs: 99.3 % / Observed criterion σ(F): 0 / Observed criterion σ(I): 0 / Redundancy: 4.8 % / Biso Wilson estimate: 14.4 Å2 / Rmerge(I) obs: 0.055 / Net I/σ(I): 32.6
Reflection shellResolution: 2.2→2.28 Å / Redundancy: 4 % / Rmerge(I) obs: 0.144 / Mean I/σ(I) obs: 10.3 / Num. unique all: 1181 / % possible all: 99.7
Reflection
*PLUS
Lowest resolution: 28.3 Å
Reflection shell
*PLUS
% possible obs: 99.7 % / Num. unique obs: 1181

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Processing

Software
NameClassification
MAR345data collection
SCALEPACKdata scaling
CNSrefinement
CNSphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB ENTRY 2YFP

2yfp


Resolution: 2.2→28.3 Å / Rfactor Rfree error: 0.006 / Data cutoff high absF: 1263802.3 / Data cutoff low absF: 0 / Isotropic thermal model: RESTRAINED / Cross valid method: THROUGHOUT / σ(F): 0 / σ(I): 0 / Stereochemistry target values: Engh & Huber
RfactorNum. reflection% reflectionSelection details
Rfree0.208 1240 10.4 %RANDOM
Rwork0.164 ---
all0.164 12153 --
obs0.164 11971 98.4 %-
Solvent computationSolvent model: FLAT MODEL / Bsol: 32.57 Å2 / ksol: 0.3133 e/Å3
Displacement parametersBiso mean: 28.2 Å2
Baniso -1Baniso -2Baniso -3
1--3.19 Å20 Å20 Å2
2---0.36 Å20 Å2
3---3.55 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.26 Å0.2 Å
Luzzati d res low-5 Å
Luzzati sigma a0.19 Å0.13 Å
Refinement stepCycle: LAST / Resolution: 2.2→28.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1852 0 0 97 1949
Refine LS restraints
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_bond_d0.009
X-RAY DIFFRACTIONc_bond_d_na
X-RAY DIFFRACTIONc_bond_d_prot
X-RAY DIFFRACTIONc_angle_d
X-RAY DIFFRACTIONc_angle_d_na
X-RAY DIFFRACTIONc_angle_d_prot
X-RAY DIFFRACTIONc_angle_deg1.3
X-RAY DIFFRACTIONc_angle_deg_na
X-RAY DIFFRACTIONc_angle_deg_prot
X-RAY DIFFRACTIONc_dihedral_angle_d26.5
X-RAY DIFFRACTIONc_dihedral_angle_d_na
X-RAY DIFFRACTIONc_dihedral_angle_d_prot
X-RAY DIFFRACTIONc_improper_angle_d0.84
X-RAY DIFFRACTIONc_improper_angle_d_na
X-RAY DIFFRACTIONc_improper_angle_d_prot
X-RAY DIFFRACTIONc_mcbond_it1.471.5
X-RAY DIFFRACTIONc_mcangle_it2.32
X-RAY DIFFRACTIONc_scbond_it2.582
X-RAY DIFFRACTIONc_scangle_it3.922.5
LS refinement shellResolution: 2.2→2.28 Å / Rfactor Rfree error: 0.022 / Total num. of bins used: 10
RfactorNum. reflection% reflection
Rfree0.26 138 11.9 %
Rwork0.195 1018 -
obs-1156 97.7 %
Xplor file
Refine-IDSerial noParam fileTopol file
X-RAY DIFFRACTION1PROTEIN_REP.PARAMPROTEIN.TOP
X-RAY DIFFRACTION2DNA-RNA_REP.PARAMDNA-RNA.TOP
X-RAY DIFFRACTION3WATER_REP.PARAMWATER.TOP
X-RAY DIFFRACTION4ION.PARAMION.TOP
X-RAY DIFFRACTION5CRO.PARAMCRO.TOP
Software
*PLUS
Name: CNS / Version: 1 / Classification: refinement
Refinement
*PLUS
σ(F): 0 / % reflection Rfree: 10.4 %
Solvent computation
*PLUS
Displacement parameters
*PLUS
Biso mean: 28.2 Å2
Refine LS restraints
*PLUS
Refine-IDTypeDev idealDev ideal target
X-RAY DIFFRACTIONc_angle_deg1.3
X-RAY DIFFRACTIONc_dihedral_angle_d
X-RAY DIFFRACTIONc_dihedral_angle_deg26.5
X-RAY DIFFRACTIONc_improper_angle_d
X-RAY DIFFRACTIONc_improper_angle_deg0.84
X-RAY DIFFRACTIONc_mcbond_it1.471.5
X-RAY DIFFRACTIONc_scbond_it2.582
X-RAY DIFFRACTIONc_mcangle_it2.32
X-RAY DIFFRACTIONc_scangle_it3.922.5
LS refinement shell
*PLUS
Rfactor Rfree: 0.26 / % reflection Rfree: 11.9 % / Rfactor Rwork: 0.195

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