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

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Basic information

Entry
Database: PDB / ID: 9ebx
TitleChimeric fluorescence biosensor formed from a lactate-binding protein and GFP
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
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: 2.42 Å
AuthorsHorwitz, S.M. / Ambarian, J.A. / 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)


Theoretical massNumber of molelcules
Total (without water)58,4411
Polymers58,4411
Non-polymers00
Water1629
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)34.350, 57.980, 67.319
Angle α, β, γ (deg.)97.82, 91.43, 104.24
Int Tables number1
Space group name H-MP1

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Components

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


Mass: 58440.969 Da / Num. of mol.: 1
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 B (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): OneShot / References: UniProt: P42212, UniProt: O24911
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / 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: 2.22 Å3/Da / Density % sol: 44.6 %
Description: Bright yellow chunky diamond-shaped crystals, 200 x 100 um
Crystal growTemperature: 296 K / Method: vapor diffusion, sitting drop / Details: magnesium chloride hexahydrate, HEPES, PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 23-ID-B / Wavelength: 0.978532 Å
DetectorType: DECTRIS EIGER X 16M / Detector: PIXEL / Date: Jul 7, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.978532 Å / Relative weight: 1
ReflectionResolution: 2.42→46.13 Å / Num. obs: 16566 / % possible obs: 86.92 % / Redundancy: 1.9 % / CC1/2: 0.999 / CC star: 1 / Rmerge(I) obs: 0.03116 / Rpim(I) all: 0.03116 / Rrim(I) all: 0.04407 / Net I/σ(I): 12.56
Reflection shellResolution: 2.42→2.507 Å / Redundancy: 1.9 % / Rmerge(I) obs: 0.3715 / Mean I/σ(I) obs: 1.75 / Num. unique obs: 1775 / CC1/2: 0.874 / CC star: 0.966 / Rpim(I) all: 0.3715 / Rrim(I) all: 0.5253 / % possible all: 92.11

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487: 000refinement
Aimless1.19data scaling
XDS0.7.15data reduction
PHASER2.8.3phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.42→46.13 Å / SU ML: 0.43 / Cross valid method: FREE R-VALUE / σ(F): 1.97 / Phase error: 37.03 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2796 1645 9.93 %
Rwork0.234 --
obs0.2386 16566 86.86 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 2.42→46.13 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3518 0 0 9 3527
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0093590
X-RAY DIFFRACTIONf_angle_d1.2424888
X-RAY DIFFRACTIONf_dihedral_angle_d15.8681205
X-RAY DIFFRACTIONf_chiral_restr0.06559
X-RAY DIFFRACTIONf_plane_restr0.015639
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.42-2.490.39911570.33951304X-RAY DIFFRACTION91
2.49-2.570.34631330.32281292X-RAY DIFFRACTION91
2.57-2.660.37331460.30121276X-RAY DIFFRACTION88
2.66-2.770.45071250.31561218X-RAY DIFFRACTION84
2.77-2.890.39741310.33811165X-RAY DIFFRACTION82
2.89-3.040.37841430.29531275X-RAY DIFFRACTION90
3.04-3.240.34131480.27461287X-RAY DIFFRACTION90
3.24-3.480.3551210.28671230X-RAY DIFFRACTION85
3.49-3.840.30321380.25291201X-RAY DIFFRACTION84
3.84-4.390.25451210.20561121X-RAY DIFFRACTION78
4.39-5.530.22981470.19051310X-RAY DIFFRACTION92
5.53-46.130.20531350.19111242X-RAY DIFFRACTION87

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