1X9Q

4m5.3 anti-fluorescein single chain antibody fragment (scFv)


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.193 
  • R-Value Work: 0.165 
  • R-Value Observed: 0.193 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Substantial energetic improvement with minimal structural perturbation in a high affinity mutant antibody

Midelfort, K.S.Hernandez, H.H.Lippow, S.M.Tidor, B.Drennan, C.L.Wittrup, K.D.

(2004) J Mol Biol 343: 685-701

  • DOI: https://doi.org/10.1016/j.jmb.2004.08.019
  • Primary Citation of Related Structures:  
    1X9Q

  • PubMed Abstract: 

    Here, we compare an antibody with the highest known engineered affinity (K(d)=270 fM) to its high affinity wild-type (K(d)=700 pM) through thermodynamic, kinetic, structural, and theoretical analyses. The 4M5.3 anti-fluorescein single chain antibody fragment (scFv) contains 14 mutations from the wild-type 4-4-20 scFv and has a 1800-fold increase in fluorescein-binding affinity. The dissociation rate is approximately 16,000 times slower in the mutant; however, this substantial improvement is offset somewhat by the association rate, which is ninefold slower in the mutant. Enthalpic contributions to binding were found by calorimetry to predominate in the differential binding free energy. The crystal structure of the 4M5.3 mutant complexed with antigen was solved to 1.5A resolution and compared with a previously solved structure of an antigen-bound 4-4-20 Fab fragment. Strikingly, the structural comparison shows little difference between the two scFv molecules (backbone RMSD of 0.6A), despite the large difference in affinity. Shape complementarity exhibits a small improvement between the variable light chain and variable heavy chain domains within the antibody, but no significant improvement in shape complementarity of the antibody with the antigen is observed in the mutant over the wild-type. Theoretical modeling calculations show electrostatic contributions to binding account for -1.2 kcal/mol to -3.5 kcal/mol of the binding free energy change, of which -1.1 kcal/mol is directly associated with the mutated residue side-chains. The electrostatic analysis reveals several mechanistic explanations for a portion of the improvement. Collectively, these data provide an example where very high binding affinity is achieved through the cumulative effect of many small structural alterations.


  • Organizational Affiliation

    Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
4m5.3 anti-fluorescein single chain antibody fragment268Homo sapiensMutation(s): 0 
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.50 Å
  • R-Value Free: 0.193 
  • R-Value Work: 0.165 
  • R-Value Observed: 0.193 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 55.78α = 90
b = 57.45β = 90
c = 79.22γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALEPACKdata scaling
XTALVIEWrefinement
REFMACrefinement

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2005-03-22
    Type: Initial release
  • Version 1.1: 2008-04-30
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2017-10-11
    Changes: Advisory, Database references, Structure summary
  • Version 1.4: 2023-08-23
    Changes: Advisory, Data collection, Database references, Derived calculations, Refinement description