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Purification, crystallization and preliminary X-ray analysis of a deletion mutant of a major buckwheat allergen.

Kezuka Y, Itagaki T, Satoh R, Teshima R, Nonaka T - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2009)

Bottom Line: A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active.The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 A, alpha = 111.92, beta = 108.91, gamma = 98.74 degrees .One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Structural Biology, School of Pharmacy, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.

ABSTRACT
A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16DeltaN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 A, alpha = 111.92, beta = 108.91, gamma = 98.74 degrees . One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

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A section at w = 0.317 of the anomalous difference Patterson map for the SeMet derivative containing the highest peak. The map was calculated using the peak data and is contoured at intervals of 0.5σ starting at 3.0σ above the mean density level.
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fig2: A section at w = 0.317 of the anomalous difference Patterson map for the SeMet derivative containing the highest peak. The map was calculated using the peak data and is contoured at intervals of 0.5σ starting at 3.0σ above the mean density level.

Mentions: We first attempted to solve the structure of rBWp16ΔN using molecular-replacement (MR) techniques. Three homology models were constructed based on the above-mentioned protein structures using MODELLER (Sali & Blundell, 1993 ▶) and were used as search models. However, no significant peaks were found in the rotation and translation functions despite exhaustive attempts and the calculated phases from the MR solutions did not give any interpretable electron density. Therefore, we prepared SeMet-labelled rBWp16ΔN and crystallized it for MAD structure determination. The purified protein yield decreased to 0.1 mg per litre of medium for derivatization. SeMet-labelled rBWp16ΔN crystals were grown under conditions I and II. Fig. 1 ▶(b) shows SeMet-labelled rBWp16ΔN crystals obtained under condition II. In the XAFS experiment a clear selenium absorption edge was monitored from a crystal, enabling us to determine the peak (0.9786 Å), edge (0.9791 Å) and remote (0.9639 Å) wavelengths. A three-wavelength MAD data set was collected to 1.60 Å resolution from a crystal obtained under condition II. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 Å, α = 111.92, β = 108.91, γ = 98.74°. One monomer was expected to be present in the unit cell based on the calculated Matthews coefficient of 1.76 Å3 Da−1 (Matthews, 1968 ▶), corresponding to a solvent content of 30%. Table 1 ▶ shows a summary of the data-collection and data-processing statistics. The peak data set showed strong peaks derived from Se atoms in an anomalous difference Patterson map contoured at 3.0σ above the mean density level. A section of the map containing the highest peak is shown in Fig. 2 ▶. Phasing calculations using SOLVE (Terwilliger & Berendzen, 1999 ▶) are now in progress.


Purification, crystallization and preliminary X-ray analysis of a deletion mutant of a major buckwheat allergen.

Kezuka Y, Itagaki T, Satoh R, Teshima R, Nonaka T - Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. (2009)

A section at w = 0.317 of the anomalous difference Patterson map for the SeMet derivative containing the highest peak. The map was calculated using the peak data and is contoured at intervals of 0.5σ starting at 3.0σ above the mean density level.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2802877&req=5

fig2: A section at w = 0.317 of the anomalous difference Patterson map for the SeMet derivative containing the highest peak. The map was calculated using the peak data and is contoured at intervals of 0.5σ starting at 3.0σ above the mean density level.
Mentions: We first attempted to solve the structure of rBWp16ΔN using molecular-replacement (MR) techniques. Three homology models were constructed based on the above-mentioned protein structures using MODELLER (Sali & Blundell, 1993 ▶) and were used as search models. However, no significant peaks were found in the rotation and translation functions despite exhaustive attempts and the calculated phases from the MR solutions did not give any interpretable electron density. Therefore, we prepared SeMet-labelled rBWp16ΔN and crystallized it for MAD structure determination. The purified protein yield decreased to 0.1 mg per litre of medium for derivatization. SeMet-labelled rBWp16ΔN crystals were grown under conditions I and II. Fig. 1 ▶(b) shows SeMet-labelled rBWp16ΔN crystals obtained under condition II. In the XAFS experiment a clear selenium absorption edge was monitored from a crystal, enabling us to determine the peak (0.9786 Å), edge (0.9791 Å) and remote (0.9639 Å) wavelengths. A three-wavelength MAD data set was collected to 1.60 Å resolution from a crystal obtained under condition II. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 Å, α = 111.92, β = 108.91, γ = 98.74°. One monomer was expected to be present in the unit cell based on the calculated Matthews coefficient of 1.76 Å3 Da−1 (Matthews, 1968 ▶), corresponding to a solvent content of 30%. Table 1 ▶ shows a summary of the data-collection and data-processing statistics. The peak data set showed strong peaks derived from Se atoms in an anomalous difference Patterson map contoured at 3.0σ above the mean density level. A section of the map containing the highest peak is shown in Fig. 2 ▶. Phasing calculations using SOLVE (Terwilliger & Berendzen, 1999 ▶) are now in progress.

Bottom Line: A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active.The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 A, alpha = 111.92, beta = 108.91, gamma = 98.74 degrees .One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Structural Biology, School of Pharmacy, Iwate Medical University, Yahaba, Iwate 028-3694, Japan.

ABSTRACT
A 16 kDa buckwheat protein (BWp16) is a major allergen responsible for immediate hypersensitivity reactions including anaphylaxis. A deletion mutant of BWp16 (rBWp16DeltaN) was overproduced and purified and was shown to be immunologically active. A three-wavelength MAD data set was collected from a crystal of selenomethionine-labelled rBWp16DeltaN. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 28.39, b = 31.54, c = 32.20 A, alpha = 111.92, beta = 108.91, gamma = 98.74 degrees . One monomer was expected to be present in the asymmetric unit based on the calculated Matthews coefficient of 1.76 A(3) Da(-1).

Show MeSH
Related in: MedlinePlus