4EEU

Crystal structure of phiLOV2.1


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.41 Å
  • R-Value Free: 0.212 
  • R-Value Work: 0.201 
  • R-Value Observed: 0.202 

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Ligand Structure Quality Assessment 


This is version 1.3 of the entry. See complete history


Literature

Structural Tuning of the Fluorescent Protein iLOV for Improved Photostability.

Christie, J.M.Hitomi, K.Arvai, A.S.Hartfield, K.A.Mettlen, M.Pratt, A.J.Tainer, J.A.Getzoff, E.D.

(2012) J Biol Chem 287: 22295-22304

  • DOI: https://doi.org/10.1074/jbc.M111.318881
  • Primary Citation of Related Structures:  
    4EEP, 4EER, 4EES, 4EET, 4EEU

  • PubMed Abstract: 

    Fluorescent proteins derived from light, oxygen, or voltage (LOV) domains offer advantages over green fluorescent protein (GFP) from their small size and efficacy under anaerobic conditions. The flavoprotein improved LOV (iLOV) was engineered from the blue light receptor phototropin as a reporter of viral infection. To inform the molecular basis for the improved, photoreversible, fluorescent properties of iLOV, we employed directed evolution and determined five LOV crystallographic structures. Comparative structural analyses between iLOV and its progenitors reveal mutation-induced constraints in the environment of the flavin mononucleotide (FMN) chromophore; in iLOV, the methyl group of Thr-394 "crowds" the FMN isoalloxazine ring, Leu-470 triggers side chain "flipping" of Leu-472, and the terminal FMN phosphate shows increased anchoring. We further engineered iLOV variants that are readily detectable in bacterial and mammalian cells due to order-of-magnitude photostability increases. Structure determination of a resulting representative photostable iLOV (phiLOV) variant reveals additional constraints on the chromophore. Aromatic residues Tyr-401 and Phe-485 in phiLOV sandwich the FMN isoalloxazine ring from both sides, whereas Ser-390 anchors the side chain of FMN-interacting Gln-489 Our combined structural and mutational results reveal that constraining the FMN fluorophore yields improved photochemical properties for iLOV and its new photostable derivative. These findings provide a framework for structural fine-tuning of LOV scaffold proteins to maximize their potential as oxygen-independent fluorescent reporters.


  • Organizational Affiliation

    Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. john.christie@glasgow.ac.uk


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Phototropin-2118Arabidopsis thalianaMutation(s): 0 
Gene Names: PHOT2CAV1KIN7NPL1At5g58140K21L19.6
EC: 2.7.11.1
UniProt
Find proteins for P93025 (Arabidopsis thaliana)
Explore P93025 
Go to UniProtKB:  P93025
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP93025
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
FMN
Query on FMN

Download Ideal Coordinates CCD File 
B [auth A]FLAVIN MONONUCLEOTIDE
C17 H21 N4 O9 P
FVTCRASFADXXNN-SCRDCRAPSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.41 Å
  • R-Value Free: 0.212 
  • R-Value Work: 0.201 
  • R-Value Observed: 0.202 
  • Space Group: P 43 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 40.967α = 90
b = 40.967β = 90
c = 130.554γ = 90
Software Package:
Software NamePurpose
HKL-2000data collection
PHASESphasing
PHENIXrefinement
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2012-05-16
    Type: Initial release
  • Version 1.1: 2012-06-06
    Changes: Database references
  • Version 1.2: 2012-07-11
    Changes: Database references
  • Version 1.3: 2023-09-13
    Changes: Data collection, Database references, Derived calculations, Refinement description