3KKW

Crystal structure of His-tagged form of PA4794 protein


Experimental Data Snapshot

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.41 Å
  • R-Value Free: 0.179 
  • R-Value Work: 0.156 
  • R-Value Observed: 0.157 

wwPDB Validation   3D Report Full Report


This is version 1.4 of the entry. See complete history


Literature

Double trouble-Buffer selection and His-tag presence may be responsible for nonreproducibility of biomedical experiments.

Majorek, K.A.Kuhn, M.L.Chruszcz, M.Anderson, W.F.Minor, W.

(2014) Protein Sci 23: 1359-1368

  • DOI: https://doi.org/10.1002/pro.2520
  • Primary Citation of Related Structures:  
    3KKW, 4M3S

  • PubMed Abstract: 

    The availability of purified and active protein is the starting point for the majority of in vitro biomedical, biochemical, and drug discovery experiments. The use of polyhistidine affinity tags has resulted in great increases of the efficiency of the protein purification process, but can negatively affect structure and/or activity measurements. Similarly, buffer molecules may perturb the conformational stability of a protein or its activity. During the determination of the structure of a Gcn5-related N-acetyltransferase (GNAT) from Pseudomonas aeruginosa (PA4794), we found that both HEPES and the polyhistidine affinity tag bind (separately) in the substrate-binding site. In the case of HEPES, the molecule induces conformational changes in the active site, but does not significantly affect enzyme activity. In contrast, the uncleaved His-tag does not induce major conformational changes but acts as a weak competitive inhibitor of peptide substrate. In two other GNAT enzymes, we observed that the presence of the His-tag had a strong influence on the activity of these proteins. The influence of protein preparation on functional studies may affect the reproducibility of experiments in other laboratories, even when changes between protocols seem at first glance to be insignificant. Moreover, the results presented here show how critical it is to adjust the experimental conditions for each protein or family of proteins, and investigate the influence of these factors on protein activity and structure, as they may significantly alter the effectiveness of functional characterization and screening methods. Thus, we show that a polyhistidine tag and the buffer molecule HEPES bind in the substrate-binding site and influence the conformation of the active site and the activity of GNAT acetyltransferases. We believe that such discrepancies can influence the reproducibility of some experiments and therefore could have a significant "ripple effect" on subsequent studies.


  • Organizational Affiliation

    Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, Virginia, 22908; Bioinformatics Laboratory, Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University, 61-614, Poznan, Poland; Midwest Center for Structural Genomics, USA; Center for Structural Genomics of Infectious Diseases (CSGID), USA.


Macromolecules
Find similar proteins by:  (by identity cutoff)  |  3D Structure
Entity ID: 1
MoleculeChains Sequence LengthOrganismDetailsImage
Putative uncharacterized protein182Pseudomonas aeruginosa PAO1Mutation(s): 0 
Gene Names: PA4794
UniProt
Find proteins for Q9HV14 (Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1))
Explore Q9HV14 
Go to UniProtKB:  Q9HV14
Entity Groups  
Sequence Clusters30% Identity50% Identity70% Identity90% Identity95% Identity100% Identity
UniProt GroupQ9HV14
Sequence Annotations
Expand
  • Reference Sequence
Small Molecules
Ligands 2 Unique
IDChains Name / Formula / InChI Key2D Diagram3D Interactions
SO4
Query on SO4

Download Ideal Coordinates CCD File 
B [auth A],
C [auth A]
SULFATE ION
O4 S
QAOWNCQODCNURD-UHFFFAOYSA-L
EDO
Query on EDO

Download Ideal Coordinates CCD File 
D [auth A]
E [auth A]
F [auth A]
G [auth A]
H [auth A]
D [auth A],
E [auth A],
F [auth A],
G [auth A],
H [auth A],
I [auth A],
J [auth A],
K [auth A],
L [auth A],
M [auth A],
N [auth A],
O [auth A],
P [auth A],
Q [auth A],
R [auth A],
S [auth A],
T [auth A]
1,2-ETHANEDIOL
C2 H6 O2
LYCAIKOWRPUZTN-UHFFFAOYSA-N
Experimental Data & Validation

Experimental Data

  • Method: X-RAY DIFFRACTION
  • Resolution: 1.41 Å
  • R-Value Free: 0.179 
  • R-Value Work: 0.156 
  • R-Value Observed: 0.157 
  • Space Group: P 21 21 2
Unit Cell:
Length ( Å )Angle ( ˚ )
a = 57.534α = 90
b = 76.145β = 90
c = 39.454γ = 90
Software Package:
Software NamePurpose
HKL-2000data collection
HKL-3000phasing
REFMACrefinement
Cootmodel building
HKL-2000data reduction
HKL-2000data scaling

Structure Validation

View Full Validation Report



Entry History 

Deposition Data

Revision History  (Full details and data files)

  • Version 1.0: 2009-11-24
    Type: Initial release
  • Version 1.1: 2011-07-13
    Changes: Version format compliance
  • Version 1.2: 2022-04-13
    Changes: Database references, Derived calculations, Structure summary
  • Version 1.3: 2023-08-09
    Changes: Data collection, Database references
  • Version 1.4: 2023-09-06
    Changes: Refinement description