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- PDB-5bt0: Switching GFP fluorescence using genetically encoded phenyl azide... -

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Basic information

Entry
Database: PDB / ID: 5bt0
TitleSwitching GFP fluorescence using genetically encoded phenyl azide chemistry through two different non-native post-translational modifications routes at the same position.
ComponentsGreen fluorescent protein
KeywordsFLUORESCENT PROTEIN / synthetic biology / photocontrol / optogenetics / unnatural amino acids / protein fluorescence / sfGFP
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 / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 2.03 Å
AuthorsHartley, A.M. / Worthy, H.L. / Reddington, S.C. / Rizkallah, P.J. / Jones, D.D.
CitationJournal: Chem Sci / Year: 2016
Title: Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle.
Authors: Hartley, A.M. / Worthy, H.L. / Reddington, S.C. / Rizkallah, P.J. / Jones, D.D.
History
DepositionJun 2, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jul 13, 2016Provider: repository / Type: Initial release
Revision 1.1May 10, 2017Group: Database references

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Green fluorescent protein
B: Green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)52,8918
Polymers52,3152
Non-polymers5766
Water6,215345
1
A: Green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,4464
Polymers26,1571
Non-polymers2883
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,4464
Polymers26,1571
Non-polymers2883
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)135.600, 135.600, 69.230
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Components on special symmetry positions
IDModelComponents
11A-401-

HOH

21A-574-

HOH

31B-568-

HOH

Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
12A
22B

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1010A2 - 64
2010B2 - 64
1020A68 - 234
2020B68 - 234

NCS ensembles :
ID
1
2

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Components

#1: Protein Green fluorescent protein /


Mass: 26157.416 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: gfp / Production host: Escherichia coli (E. coli) / References: UniProt: A0A059PIQ0
#2: Chemical
ChemComp-SO4 / SULFATE ION / Sulfate


Mass: 96.063 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 345 / Source method: isolated from a natural source / Formula: H2O

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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: 3.04 Å3/Da / Density % sol: 59.56 %
Crystal growTemperature: 297 K / Method: vapor diffusion, sitting drop / pH: 8.5 / Details: 50 mM MMT, 2.5 M (NH4)2SO4

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I04-1 / Wavelength: 0.92 Å
DetectorType: DECTRIS PILATUS 2M / Detector: PIXEL / Date: Nov 13, 2012
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.92 Å / Relative weight: 1
ReflectionResolution: 2.03→42.88 Å / Num. obs: 42237 / % possible obs: 100 % / Redundancy: 14.2 % / CC1/2: 0.998 / Rmerge(I) obs: 0.107 / Rpim(I) all: 0.03 / Net I/σ(I): 16.7 / Num. measured all: 600948
Reflection shell

Diffraction-ID: 1 / Rejects: 0

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allCC1/2Rpim(I) all% possible all
2.03-2.0813.80.7523.94238330820.8860.209100
9.08-42.8813.40.04339.775355610.9990.01299.1

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation4.19 Å42.88 Å
Translation4.19 Å42.88 Å

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Processing

Software
NameVersionClassification
Aimless0.1.27data scaling
PHASER2.5.2phasing
REFMAC5.8.0073refinement
PDB_EXTRACT3.15data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.03→42.88 Å / Cor.coef. Fo:Fc: 0.963 / Cor.coef. Fo:Fc free: 0.948 / SU B: 7.399 / SU ML: 0.107 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.147 / ESU R Free: 0.135 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2042 2133 5.1 %RANDOM
Rwork0.1716 ---
obs0.1733 40050 99.98 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 169.65 Å2 / Biso mean: 39.448 Å2 / Biso min: 20.4 Å2
Baniso -1Baniso -2Baniso -3
1--0.05 Å20 Å20 Å2
2---0.05 Å20 Å2
3---0.1 Å2
Refinement stepCycle: final / Resolution: 2.03→42.88 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3680 0 30 345 4055
Biso mean--118.64 41.49 -
Num. residues----462
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0193811
X-RAY DIFFRACTIONr_bond_other_d0.0060.023567
X-RAY DIFFRACTIONr_angle_refined_deg2.0291.9785155
X-RAY DIFFRACTIONr_angle_other_deg1.25238234
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.6525462
X-RAY DIFFRACTIONr_dihedral_angle_2_deg34.25425.028181
X-RAY DIFFRACTIONr_dihedral_angle_3_deg15.92615652
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.4731516
X-RAY DIFFRACTIONr_chiral_restr0.1340.2561
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.0214293
X-RAY DIFFRACTIONr_gen_planes_other0.0050.02875
X-RAY DIFFRACTIONr_mcbond_it1.8911.7531848
X-RAY DIFFRACTIONr_mcbond_other1.8171.751847
X-RAY DIFFRACTIONr_mcangle_it2.8862.6032307
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A31420.11
12B31420.11
21A88190.13
22B88190.13
LS refinement shellResolution: 2.03→2.083 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.277 164 -
Rwork0.25 2907 -
all-3071 -
obs--100 %
Refinement TLS params.

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

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.87890.79140.59694.0789-0.64551.79180.26880.19470.0449-0.3819-0.23780.3194-0.00070.1444-0.03090.13110.0433-0.08890.037-0.01220.106222.99331.42616.874
24.08511.288-0.70642.3356-0.65972.23940.05940.0651-0.1968-0.0637-0.1362-0.29050.0575-0.02570.07690.0084-0.0015-0.0120.05640.09510.198358.24241.62623.691
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 231
2X-RAY DIFFRACTION2B1 - 231

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