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Purification, crystallization and preliminary crystallographic analysis of the minor pilin FctB from Streptococcus
Christian Linke1, Paul G Young, Hae Joo Kang
1School of Biological Sciences, University of Auckland, Auckland, New Zealand.
Abstract:
The minor pilin FctB is an integral part of the pilus assembly expressed by Streptococcus pyogenes. Since it is located at the cell wall, it can be hypothesized that it functions as a cell-wall anchor for the streptococcal pilus. In order to elucidate its structure, the genes for FctB from the S. pyogenes strains 90/306S and SF370 were cloned for overexpression in Escherichia coli. FctB from strain 90/306S was crystallized by the sitting-drop vapour-diffusion method using sodium citrate as a precipitant. The hexagonal FctB crystals belonged to space group P6(1) or P6(5), with unit-cell parameters a = b = 95.15, c = 100.25 A, and diffracted to 2.9 A resolution.
Insights
The minor pilin FctB from Streptococcus pyogenes, potentially a cell-wall anchor, was structurally analyzed. Researchers successfully crystallized and determined the resolution of FctB, aiding in understanding pilus assembly.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- The minor pilin FctB is a key component of pilus assembly in Streptococcus pyogenes.
- Its location at the cell wall suggests a role as a structural anchor for the streptococcal pilus.
Purpose of the Study:
- To elucidate the structure of the FctB protein.
- To understand the role of FctB in pilus assembly and cell-wall anchoring in Streptococcus pyogenes.
Main Methods:
- Genes for FctB from two S. pyogenes strains were cloned and overexpressed in Escherichia coli.
- FctB protein was purified and crystallized using the sitting-drop vapour-diffusion method with sodium citrate.
- X-ray diffraction data was collected to determine crystal structure and resolution.
Main Results:
- Hexagonal FctB crystals were obtained, belonging to space group P6(1) or P6(5).
- Unit-cell parameters were determined as a = b = 95.15 Å and c = 100.25 Å.
- The crystals diffracted X-rays to a resolution of 2.9 Å.
Conclusions:
- The structural determination of FctB provides insights into its function as a potential cell-wall anchor.
- This research lays the foundation for further studies on streptococcal pilus biogenesis and function.
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