6BJZ

Crystal Structure of the Fab fragment of humanized 5c8 antibody containing the fluorescent non-canonical amino acid L-(7-hydroxycoumarin-4-yl)ethylglycine at pH 5.5


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.45 Å
  • R-Value Free: 0.170 
  • R-Value Work: 0.122 
  • R-Value Observed: 0.124 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Structural Insights into How Protein Environments Tune the Spectroscopic Properties of a Noncanonical Amino Acid Fluorophore.

Henderson, J.N.Simmons, C.R.Fahmi, N.E.Jeffs, J.W.Borges, C.R.Mills, J.H.

(2020) Biochemistry 59: 3401-3410

  • DOI: https://doi.org/10.1021/acs.biochem.0c00474
  • Primary Citation of Related Structures:  
    6BJZ, 6W4W, 6W5A, 6W9G

  • PubMed Abstract: 

    Genetically encoded fluorescent noncanonical amino acids (fNCAAs) could be used to develop novel fluorescent sensors of protein function. Previous efforts toward this goal have been limited by the lack of extensive physicochemical and structural characterizations of protein-based sensors containing fNCAAs. Here, we report the steady-state spectroscopic properties and first structural analyses of an fNCAA-containing Fab fragment of the 5c8 antibody, which binds human CD40L. A previously reported 5c8 variant in which the light chain residue Ile L 98 is replaced with the fNCAA l-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA) exhibits a 1.7-fold increase in fluorescence upon antigen binding. Determination and comparison of the apparent p K a s of 7-HCAA in the unbound and bound forms indicate that the observed increase in fluorescence is not the result of perturbations in p K a . Crystal structures of the fNCAA-containing Fab in the apo and bound forms reveal interactions between the 7-HCAA side chain and surrounding residues that are disrupted upon antigen binding. This structural characterization not only provides insight into the manner in which protein environments can modulate the fluorescence properties of 7-HCAA but also could serve as a starting point for the rational design of new fluorescent protein-based reporters of protein function.


  • Organizational Affiliation

    The Biodesign Center for Molecular Design and Biomimetics, Arizona State University, Tempe, Arizona 85287, United States.


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
5c8 Fab Heavy chainA [auth H]226Homo sapiensMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
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  • Reference Sequence
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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
5c8 Fab Light chainB [auth L]218Homo sapiensMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
Expand
  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.45 Å
  • R-Value Free: 0.170 
  • R-Value Work: 0.122 
  • R-Value Observed: 0.124 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 90.813α = 90
b = 59.911β = 96.48
c = 73.459γ = 90
Software Package:
Software NamePurpose
HKL-2000data scaling
PHASERphasing
REFMACrefinement
PDB_EXTRACTdata extraction
HKL-2000data reduction

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2018-11-14
    Type: Initial release
  • Version 1.1: 2020-12-23
    Changes: Database references
  • Version 1.2: 2023-10-04
    Changes: Data collection, Database references, Refinement description
  • Version 1.3: 2023-11-15
    Changes: Data collection, Derived calculations