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- PDB-9ebw: Chimeric fluorescence biosensor formed from a lactate-binding pro... -

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Basic information

Entry
Database: PDB / ID: 9ebw
TitleChimeric fluorescence biosensor formed from a lactate-binding protein and GFP, bound to lactate
ComponentsGreen fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)
KeywordsFLUORESCENT PROTEIN / Biosensor / Binding protein / Chimera
Function / homology
Function and homology information


bioluminescence / generation of precursor metabolites and energy / chemotaxis / signal transduction / membrane
Similarity search - Function
Methyl-accepting chemotaxis protein (MCP) signalling domain / Methyl-accepting chemotaxis protein (MCP) signalling domain / Bacterial chemotaxis sensory transducers domain profile. / Methyl-accepting chemotaxis-like domains (chemotaxis sensory transducer). / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein
Similarity search - Domain/homology
LACTIC ACID / Methyl-accepting chemotaxis transducer (TlpC) / Green fluorescent protein
Similarity search - Component
Biological speciesAequorea victoria (jellyfish)
Helicobacter pylori (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.78 Å
AuthorsHorwitz, S.M. / Ambarian, J.A. / Jones, R. / Waidmann, L. / Davis, K.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01GM124038 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)F31CA254162 United States
National Science Foundation (NSF, United States)1937971 United States
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2025
Title: State-dependent motion of a genetically encoded fluorescent biosensor.
Authors: Rosen, P.C. / Horwitz, S.M. / Brooks, D.J. / Kim, E. / Ambarian, J.A. / Waidmann, L. / Davis, K.M. / Yellen, G.
History
DepositionNov 13, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Green fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)
B: Green fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)
C: Green fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)
D: Green fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)234,1288
Polymers233,7644
Non-polymers3644
Water2,144119
1
A: Green fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)
B: Green fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)117,1565
Polymers116,8822
Non-polymers2743
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area5570 Å2
ΔGint-27 kcal/mol
Surface area41310 Å2
MethodPISA
2
C: Green fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)
hetero molecules

D: Green fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)


Theoretical massNumber of molelcules
Total (without water)116,9723
Polymers116,8822
Non-polymers901
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation1_655x+1,y,z1
Buried area4800 Å2
ΔGint-29 kcal/mol
Surface area40040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)90.878, 109.803, 123.711
Angle α, β, γ (deg.)90.00, 110.14, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein
Green fluorescent protein,Methyl-accepting chemotaxis transducer (TlpC)


Mass: 58440.969 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: mTurquoise GFP (residues 10-153 and 428-515) attached to lactate binding protein TlpC (residues 159-421) by two separate linkers, one from residues 154-158 and another from residues 425-427
Source: (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Helicobacter pylori (bacteria)
Gene: GFP, HP_0082 / Strain: 26695 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): OneShot / References: UniProt: P42212, UniProt: O24911
#2: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H8O3
#3: Chemical ChemComp-LAC / LACTIC ACID


Mass: 90.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C3H6O3 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 119 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
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: 2.5 Å3/Da / Density % sol: 47.4 % / Description: Green needles, 100 x 25 uM
Crystal growTemperature: 296 K / Method: vapor diffusion, sitting drop / Details: citric acid, PEG 6000, 1,3-propanediol

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-1 / Wavelength: 0.920119 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Mar 3, 2024
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.920119 Å / Relative weight: 1
ReflectionResolution: 2.78→45.94 Å / Num. obs: 55034 / % possible obs: 95.44 % / Redundancy: 4.6 % / CC1/2: 0.992 / CC star: 0.998 / Rmerge(I) obs: 0.1481 / Rpim(I) all: 0.07007 / Net I/σ(I): 7.4
Reflection shellResolution: 2.78→2.88 Å / Rmerge(I) obs: 0.6888 / Mean I/σ(I) obs: 1.82 / Num. unique obs: 5330 / CC1/2: 0.826 / CC star: 0.951 / Rpim(I) all: 0.3447 / Rrim(I) all: 0.7748

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDS1.19data reduction
Aimless0.7.15data scaling
PHASER2.8.3phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.78→45.94 Å / SU ML: 0.45 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 34.05 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2848 1996 3.63 %
Rwork0.2422 --
obs0.2437 54913 95.34 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.78→45.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14571 0 24 119 14714
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01149001
X-RAY DIFFRACTIONf_angle_d1.35620240
X-RAY DIFFRACTIONf_dihedral_angle_d16.6845170
X-RAY DIFFRACTIONf_chiral_restr0.0742286
X-RAY DIFFRACTIONf_plane_restr0.0272628
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.78-2.850.42051330.35523534X-RAY DIFFRACTION89
2.85-2.930.35481420.30183858X-RAY DIFFRACTION98
2.93-3.010.31431420.28313884X-RAY DIFFRACTION98
3.01-3.110.34351500.26373862X-RAY DIFFRACTION98
3.11-3.220.29481470.25253856X-RAY DIFFRACTION98
3.22-3.350.33761410.25443844X-RAY DIFFRACTION97
3.35-3.50.31581470.26023812X-RAY DIFFRACTION97
3.5-3.690.29171350.23783813X-RAY DIFFRACTION96
3.69-3.920.30551450.24243799X-RAY DIFFRACTION96
3.92-4.220.2831420.22343783X-RAY DIFFRACTION95
4.22-4.640.24151470.20443731X-RAY DIFFRACTION94
4.64-5.310.2231420.20613681X-RAY DIFFRACTION93
5.31-6.690.27521480.24973750X-RAY DIFFRACTION94
6.69-100.26391350.2453710X-RAY DIFFRACTION91

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