1P4B

Three-Dimensional Structure Of a Single Chain Fv Fragment Complexed With The peptide GCN4(7P-14P).


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 2.35 Å
  • R-Value Free: 0.300 
  • R-Value Work: 0.219 
  • R-Value Observed: 0.230 

wwPDB Validation   3D Report Full Report


This is version 1.3 of the entry. See complete history


Literature

Directed in Vitro Evolution and Crystallographic Analysis of a Peptide-binding Single Chain Antibody Fragment (scFv) with Low Picomolar Affinity.

Zahnd, C.Spinelli, S.Amstutz, P.Cambillau, C.

(2004) J Biol Chem 279: 18870-18877

  • DOI: https://doi.org/10.1074/jbc.M309169200
  • Primary Citation of Related Structures:  
    1P4B, 1P4I

  • PubMed Abstract: 

    We generated a single chain Fv fragment of an antibody (scFv) with a binding affinity of about 5 pm to a short peptide by applying rigorous directed evolution. Starting from a high affinity peptide binder, originally obtained by ribosome display from a murine library, we generated libraries of mutants with error-prone PCR and DNA shuffling and applied off-rate selection by using ribosome display. Crystallographic analysis of the scFv in its antigen-bound and free state showed that only few mutations, which do not make direct contact to the antigen, lead to a 500-fold affinity improvement over its potential germ line precursor. These results suggest that the affinity optimization of very high affinity binders is achieved by modulating existing interactions via subtle changes in the framework rather than by introducing new contacts. Off-rate selection in combination with ribosome display can evolve binders to the low picomolar affinity range even for peptide targets.


  • Organizational Affiliation

    Biochemisches Institut der Universität Zürich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.


Macromolecules
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Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Antibody Variable light chainA [auth L]135Mus musculusMutation(s): 0 
UniProt
Find proteins for P01723 (Mus musculus)
Explore P01723 
Go to UniProtKB:  P01723
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UniProt GroupP01723
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  • Reference Sequence
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Entity ID: 2
MoleculeChains Sequence LengthOrganismDetailsImage
Antibody Variable heavy chainB [auth H]124Mus musculusMutation(s): 0 
UniProt
Find proteins for P01820 (Mus musculus)
Explore P01820 
Go to UniProtKB:  P01820
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UniProt GroupP01820
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  • Reference Sequence

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Entity ID: 3
MoleculeChains Sequence LengthOrganismDetailsImage
GCN4(7P-14P) peptideC [auth P]12N/AMutation(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: 2.35 Å
  • R-Value Free: 0.300 
  • R-Value Work: 0.219 
  • R-Value Observed: 0.230 
  • Space Group: P 1 21 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 37.238α = 90
b = 36.291β = 90.5
c = 84.455γ = 90
Software Package:
Software NamePurpose
DENZOdata reduction
SCALAdata scaling
AMoREphasing
CNSrefinement
CCP4data scaling

Structure Validation

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Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2004-05-04
    Type: Initial release
  • Version 1.1: 2008-04-29
    Changes: Version format compliance
  • Version 1.2: 2011-07-13
    Changes: Version format compliance
  • Version 1.3: 2023-08-16
    Changes: Data collection, Database references, Refinement description