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

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Basic information

Entry
Database: PDB / ID: 5btt
TitleSwitching GFP fluorescence using genetically encoded phenyl azide chemistry through two different non-native post-translational modifications routes at the same position.
Components(Green fluorescent protein) x 2
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: 2.14 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 3, 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

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)52,94915
Polymers51,7082
Non-polymers1,24113
Water4,774265
1
A: Green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,4267
Polymers25,8541
Non-polymers5726
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)26,5238
Polymers25,8541
Non-polymers6687
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)135.140, 135.140, 69.560
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-511-

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 - 231
2020B68 - 231

NCS ensembles :
ID
1
2

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Components

#1: Protein Green fluorescent protein


Mass: 25854.080 Da / Num. of mol.: 1
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: Protein Green fluorescent protein


Mass: 25854.080 Da / Num. of mol.: 1
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
#3: Chemical
ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: SO4
#4: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 265 / 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: 3.09 Å3/Da / Density % sol: 60.23 %
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: 2.14→56.24 Å / Num. obs: 36124 / % possible obs: 100 % / Redundancy: 14.6 % / CC1/2: 0.999 / Rmerge(I) obs: 0.081 / Rpim(I) all: 0.022 / Net I/σ(I): 22.9 / Num. measured all: 525718
Reflection shell

Diffraction-ID: 1 / Rejects: _

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured allNum. unique allCC1/2Rpim(I) all% possible all
2.14-2.214.40.71443780626210.9160.193100
9.57-56.2413.30.03664.864694870.9990.0199.6

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Phasing

PhasingMethod: molecular replacement
Phasing MRModel details: Phaser MODE: MR_AUTO
Highest resolutionLowest resolution
Rotation4.19 Å56.24 Å
Translation4.19 Å56.24 Å

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Processing

Software
NameVersionClassification
Aimless0.1.27data scaling
PHASER2.5.1phasing
REFMAC5.8.0073refinement
PDB_EXTRACT3.15data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.14→56.24 Å / Cor.coef. Fo:Fc: 0.962 / Cor.coef. Fo:Fc free: 0.948 / SU B: 7.737 / SU ML: 0.105 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.172 / ESU R Free: 0.154 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : WITH TLS ADDED
RfactorNum. reflection% reflectionSelection details
Rfree0.2066 1801 5 %RANDOM
Rwork0.1689 ---
obs0.1707 34272 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: 150.41 Å2 / Biso mean: 42.408 Å2 / Biso min: 22.8 Å2
Baniso -1Baniso -2Baniso -3
1--0.19 Å20 Å20 Å2
2---0.19 Å20 Å2
3---0.39 Å2
Refinement stepCycle: final / Resolution: 2.14→56.24 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3644 0 67 265 3976
Biso mean--109.68 44.88 -
Num. residues----456
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0180.0193807
X-RAY DIFFRACTIONr_bond_other_d0.0060.023552
X-RAY DIFFRACTIONr_angle_refined_deg2.0591.9835150
X-RAY DIFFRACTIONr_angle_other_deg1.2238201
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.3985456
X-RAY DIFFRACTIONr_dihedral_angle_2_deg32.03324.944180
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.10115647
X-RAY DIFFRACTIONr_dihedral_angle_4_deg15.9351516
X-RAY DIFFRACTIONr_chiral_restr0.1190.2565
X-RAY DIFFRACTIONr_gen_planes_refined0.0110.0214248
X-RAY DIFFRACTIONr_gen_planes_other0.0050.02866
X-RAY DIFFRACTIONr_mcbond_it2.192.2641827
X-RAY DIFFRACTIONr_mcbond_other2.1862.2631826
X-RAY DIFFRACTIONr_mcangle_it3.0993.3742282
Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Rms dev position: 0.11 Å / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumber
11A3151
12B3151
21A8925
22B8925
LS refinement shellResolution: 2.14→2.196 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.238 137 -
Rwork0.206 2479 -
all-2616 -
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)
13.39731.3071-0.15012.4421-0.41412.30210.02690.0192-0.1661-0.0743-0.0801-0.19440.0882-0.22980.05320.0130.0076-0.00750.11970.04460.1013-9.4767-26.4596-10.9677
22.41010.66870.10493.6206-0.66091.63440.15620.09590.0733-0.1994-0.14210.2056-0.11510.0844-0.01410.09370.0304-0.05230.0209-0.02090.0496-44.5251-36.0363-18.0302
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A1 - 250
2X-RAY DIFFRACTION2B1 - 250

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