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Updated: Jul 3, 2026
From Constructs to Crystals – Towards Structure Determination of β-barrel Outer Membrane Proteins
Published on: July 4, 2016
Cloning, purification and preliminary crystallographic analysis of a putative DNA-binding membrane protein, YmfM,
Ling Xu1, Svetlana E Sedelnikova, Patrick J Baker
1Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN, England.
Abstract:
The Staphylococcus aureus protein YmfM contains a helix-turn-helix motif and is thought to be a putative DNA-binding protein which is associated with the membrane through a C-terminal hydrophobic transmembrane anchor. Truncation of the protein by the removal of this C-terminal hydrophobic segment has enabled the overexpression of a soluble domain of S. aureus YmfM (DeltaYmfM) in Escherichia coli, which has been purified and subsequently crystallized. Crystals of DeltaYmfM diffract to beyond 1.0 A resolution and belong to one of the pair of enantiomorphic tetragonal space groups P4(1)2(1)2 or P4(3)2(1)2, with unit-cell parameters a = b = 45.5, c = 72.9 A and one molecule in the asymmetric unit. The crystals of DeltaYmfM have an unusually low V(M) of 1.6 A(3) Da(-1), which is one of the lowest values observed for any protein to date. A full structure determination is under way in order to provide insights into the function of this protein.
Insights
Researchers crystallized a soluble domain of Staphylococcus aureus YmfM protein (DeltaYmfM). This structural study aims to uncover the DNA-binding protein's function.
Area of Science:
- Structural biology
- Microbiology
- Biochemistry
Background:
- Staphylococcus aureus protein YmfM possesses a helix-turn-helix motif, suggesting DNA-binding capabilities.
- YmfM is associated with the cell membrane via a C-terminal hydrophobic transmembrane anchor.
Purpose of the Study:
- To obtain a soluble, overexpressed domain of S. aureus YmfM (DeltaYmfM) for structural analysis.
- To crystallize DeltaYmfM and determine its crystal structure to elucidate its function.
Main Methods:
- Truncation of the C-terminal hydrophobic segment to create a soluble protein domain (DeltaYmfM).
- Overexpression of DeltaYmfM in Escherichia coli and subsequent purification.
- Crystallization of DeltaYmfM and assessment of diffraction resolution and crystal space group.
Main Results:
- Crystals of DeltaYmfM diffract beyond 1.0 A resolution.
- The crystals belong to enantiomorphic tetragonal space groups P4(1)2(1)2 or P4(3)2(1)2.
- DeltaYmfM crystals exhibit a low V(M) value (1.6 A(3) Da(-1)), indicating efficient crystal packing.
Conclusions:
- The successful crystallization of DeltaYmfM provides a foundation for detailed structural determination.
- Understanding the structure of DeltaYmfM is crucial for gaining insights into the function of Staphylococcus aureus YmfM.
