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Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens.

Ye F, Wang C, Fu Q, Zhang LH, Gao YG - Acta Crystallogr F Struct Biol Commun (2015)

Bottom Line: Both SghA and SghR form dimers in solution.The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively.Data were collected and processed, and the crystallographic parameters were within acceptable ranges.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.

ABSTRACT
Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen-host interaction.

No MeSH data available.


Related in: MedlinePlus

Diffraction patterns of SghA (a) and SghR (b). The right panel is a magnification of the boxed region. The resolution rings were generated using ADXV (http://www.scripps.edu/~arvai/adxv.html).
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fig4: Diffraction patterns of SghA (a) and SghR (b). The right panel is a magnification of the boxed region. The resolution rings were generated using ADXV (http://www.scripps.edu/~arvai/adxv.html).

Mentions: Crystallization screening and further optimization led to decent plate-shaped and rod-shaped crystals of SghA and SghR, respectively (Fig. 3 ▸). Both crystals were harvested with thorough washing with their reservoir solutions. Subsequent SDS–PAGE separation and mass-spectral identification were performed, and the results clearly indicated the crystallized macromolecules were the target proteins SghA and SghR, respectively. Therefore, data collection was carried out and the collected data sets were processed to acceptable Rmerge, completeness and 〈I/σ(I)〉 values in the highest resolution bin (Fig. 4 ▸ and Table 4 ▸). Both the SghA and the SghR crystals belonged to space group P212121. The data from the SghA crystals were processed to 1.9 Å resolution, with unit-cell parameters a = 64.2, b = 80.4, c = 184.62 Å, whereas the data from the SghR crystals were processed to 2.1 Å resolution, with unit-cell parameters a = 35.54, b = 119.45, c = 121.94 Å (Table 1 ▸). VM calculations indicated that there are two molecules in the asymmetric unit. On the basis of our molecular-weight calibration results, it appears that the crystals of both proteins contained a dimer in the asymmetric unit. Further model building and structure refinement are ongoing for both structures.


Cloning, expression, purification and crystallization of a pair of novel virulence factors, SghA and SghR, from Agrobacterium tumefaciens.

Ye F, Wang C, Fu Q, Zhang LH, Gao YG - Acta Crystallogr F Struct Biol Commun (2015)

Diffraction patterns of SghA (a) and SghR (b). The right panel is a magnification of the boxed region. The resolution rings were generated using ADXV (http://www.scripps.edu/~arvai/adxv.html).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4555920&req=5

fig4: Diffraction patterns of SghA (a) and SghR (b). The right panel is a magnification of the boxed region. The resolution rings were generated using ADXV (http://www.scripps.edu/~arvai/adxv.html).
Mentions: Crystallization screening and further optimization led to decent plate-shaped and rod-shaped crystals of SghA and SghR, respectively (Fig. 3 ▸). Both crystals were harvested with thorough washing with their reservoir solutions. Subsequent SDS–PAGE separation and mass-spectral identification were performed, and the results clearly indicated the crystallized macromolecules were the target proteins SghA and SghR, respectively. Therefore, data collection was carried out and the collected data sets were processed to acceptable Rmerge, completeness and 〈I/σ(I)〉 values in the highest resolution bin (Fig. 4 ▸ and Table 4 ▸). Both the SghA and the SghR crystals belonged to space group P212121. The data from the SghA crystals were processed to 1.9 Å resolution, with unit-cell parameters a = 64.2, b = 80.4, c = 184.62 Å, whereas the data from the SghR crystals were processed to 2.1 Å resolution, with unit-cell parameters a = 35.54, b = 119.45, c = 121.94 Å (Table 1 ▸). VM calculations indicated that there are two molecules in the asymmetric unit. On the basis of our molecular-weight calibration results, it appears that the crystals of both proteins contained a dimer in the asymmetric unit. Further model building and structure refinement are ongoing for both structures.

Bottom Line: Both SghA and SghR form dimers in solution.The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively.Data were collected and processed, and the crystallographic parameters were within acceptable ranges.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.

ABSTRACT
Two proteins, SghA and SghR, which were recently identified and characterized as novel bacterial virulence factors regulating the infection of plant hosts by Agrobacterium, were cloned, overexpressed and purified with high yield. Both SghA and SghR form dimers in solution. The purified SghA and SghR were crystallized and the crystals diffracted to 1.9 and 2.1 Å resolution, respectively. Data were collected and processed, and the crystallographic parameters were within acceptable ranges. These results will help in the determination of their structures in order to uncover the molecular mechanism of how these two proteins together control the release of plant defence signals against agrobacteria during pathogen-host interaction.

No MeSH data available.


Related in: MedlinePlus