2BSW

Crystal structure of a glyphosate-N-acetyltransferase obtained by DNA shuffling.


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.63 Å
  • R-Value Free: 0.213 
  • R-Value Work: 0.179 
  • R-Value Observed: 0.181 

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Ligand Structure Quality Assessment 


This is version 1.2 of the entry. See complete history


Literature

DNA Shuffling as a Tool for Protein Crystallization.

Keenan, R.J.Siehl, D.L.Gorton, R.Castle, L.A.

(2005) Proc Natl Acad Sci U S A 102: 8887

  • DOI: https://doi.org/10.1073/pnas.0502497102
  • Primary Citation of Related Structures:  
    2BSW

  • PubMed Abstract: 

    The success of structural studies performed on an individual target in small scale or on many targets in the system-wide scale of structural genomics depends critically on three parameters: (i) obtaining an expression system capable of producing large quantities of the macromolecule(s) of interest, (ii) purifying this material in soluble form, and (iii) obtaining diffraction-quality crystals suitable for x-ray analysis. The attrition rate caused by these constraints is often quite high. Here, we present a strategy that addresses each of these three parameters simultaneously. Using DNA shuffling to introduce functional sequence variability into a protein of interest, we screened crude lysate supernatants for soluble variants that retain enzymatic activity. Crystallization trials performed on three WT and eight shuffled enzymes revealed two variants that crystallized readily. One of these was used to determine the high-resolution structure of the enzyme by x-ray analysis. The sequence diversity introduced through shuffling efficiently samples crystal packing space by modifying the surface properties of the enzyme. The approach demonstrated here does not require guidance as to the type of mutation necessary for improvements in expression, solubility, or crystallization. The method is scaleable and can be applied in situations where a single protein is being studied or in high-throughput structural genomics programs. Furthermore, it should be readily applied to structural studies of soluble proteins, membrane proteins, and macromolecular complexes.


  • Organizational Affiliation

    Pioneer Hi-Bred International, Inc., Verdia Campus, 700A Bay Road, Redwood City, CA 94063, USA. [email protected]


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
GLYPHOSATE N-ACETYLTRANSFERASE146synthetic constructMutation(s): 0 
UniProt
Find proteins for Q65LG7 (Bacillus licheniformis (strain ATCC 14580 / DSM 13 / JCM 2505 / CCUG 7422 / NBRC 12200 / NCIMB 9375 / NCTC 10341 / NRRL NRS-1264 / Gibson 46))
Explore Q65LG7 
Go to UniProtKB:  Q65LG7
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ65LG7
Sequence Annotations
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  • Reference Sequence
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.63 Å
  • R-Value Free: 0.213 
  • R-Value Work: 0.179 
  • R-Value Observed: 0.181 
  • Space Group: C 1 2 1
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 69.323α = 90
b = 49.393β = 103.47
c = 46.492γ = 90
Software Package:
Software NamePurpose
REFMACrefinement
MOSFLMdata reduction
SCALEPACKdata scaling
CCP4phasing

Structure Validation

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Ligand Structure Quality Assessment 


Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2005-06-08
    Type: Initial release
  • Version 1.1: 2015-10-14
    Changes: Derived calculations, Non-polymer description, Other, Source and taxonomy, Version format compliance
  • Version 1.2: 2024-11-13
    Changes: Data collection, Database references, Derived calculations, Other, Structure summary