RCSB PDB - 5RGE: Crystal Structure of Kemp Eliminase HG3.17 with bound transition state analog, 277K

 5RGE

Crystal Structure of Kemp Eliminase HG3.17 with bound transition state analog, 277K


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.77 Å
  • R-Value Free: 0.179 
  • R-Value Work: 0.150 

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Ligand Structure Quality Assessment 

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Literature

Ensemble-based enzyme design can recapitulate the effects of laboratory directed evolution in silico.

Broom, A.Rakotoharisoa, R.V.Thompson, M.C.Zarifi, N.Nguyen, E.Mukhametzhanov, N.Liu, L.Fraser, J.S.Chica, R.A.

(2020) Nat Commun 11: 4808-4808

  • DOI: https://doi.org/10.1038/s41467-020-18619-x
  • Primary Citation of Related Structures:  
    5RG4, 5RG5, 5RG6, 5RG7, 5RG8, 5RG9, 5RGA, 5RGB, 5RGC, 5RGD, 5RGE, 5RGF

  • PubMed Abstract: 

    The creation of artificial enzymes is a key objective of computational protein design. Although de novo enzymes have been successfully designed, these exhibit low catalytic efficiencies, requiring directed evolution to improve activity. Here, we use room-temperature X-ray crystallography to study changes in the conformational ensemble during evolution of the designed Kemp eliminase HG3 (k cat /K M 146 M -1 s -1 ). We observe that catalytic residues are increasingly rigidified, the active site becomes better pre-organized, and its entrance is widened. Based on these observations, we engineer HG4, an efficient biocatalyst (k cat /K M 103,000 M -1 s -1 ) containing key first and second-shell mutations found during evolution. HG4 structures reveal that its active site is pre-organized and rigidified for efficient catalysis. Our results show how directed evolution circumvents challenges inherent to enzyme design by shifting conformational ensembles to favor catalytically-productive sub-states, and suggest improvements to the design methodology that incorporate ensemble modeling of crystallographic data.


  • Organizational Affiliation

    Department of Chemistry and Biomolecular Sciences, University of Ottawa, 10 Marie Curie, Ottawa, ON, K1N 6N5, Canada.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Kemp Eliminase HG3318Thermoascus aurantiacusMutation(s): 26 
EC: 3.2.1.8
UniProt
Find proteins for P23360 (Thermoascus aurantiacus)
Explore P23360 
Go to UniProtKB:  P23360
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupP23360
Sequence Annotations
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  • Reference Sequence
Small Molecules
Ligands 1 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
6NT (Subject of Investigation/LOI)
Query on 6NT

Download Ideal Coordinates CCD File 
B [auth A]6-NITROBENZOTRIAZOLE
C6 H4 N4 O2
AOCDQWRMYHJTMY-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.77 Å
  • R-Value Free: 0.179 
  • R-Value Work: 0.150 
  • Space Group: P 21 21 21
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 51.28α = 90
b = 58.13β = 90
c = 92.48γ = 90
Software Package:
Software NamePurpose
PHENIXrefinement
xia2data scaling
XDSdata reduction
PHASERphasing
PDB_EXTRACTdata extraction

Structure Validation

View Full Validation Report



Ligand Structure Quality Assessment 

Created with Raphaël 2.3.0Worse 01 BetterLigand structure goodness of fit to experimental dataBest fitted 6NTClick on this verticalbar to view details

Entry History & Funding Information

Deposition Data


Funding OrganizationLocationGrant Number
Natural Sciences and Engineering Research Council of CanadaCanada--

Revision History  (Full details and data files)

  • Version 1.0: 2020-07-22
    Type: Initial release
  • Version 1.1: 2020-12-02
    Changes: Database references
  • Version 1.2: 2021-05-12
    Changes: Structure summary
  • Version 1.3: 2024-03-06
    Changes: Data collection, Database references