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Crystallization and preliminary X-ray crystallographic analysis of Mirabilis antiviral protein
Abstract:
Mirabilis antiviral protein is a single-chain ribosome-inactivating protein purified from the tuberous root of Mirabilis jalapa L. We obtained several forms of crystals of the protein by the hanging drop vapor diffusion method, but most of these crystals were not suitable for X-ray crystallography. After refining the growth conditions, crystals of crystallographic quality were grown in 20-microliters droplets of an equi-volume mixture of 1.5% (w/v) protein solution and a reservoir solution containing 49 to 50% (w/v) ammonium sulfate and 50 mM-ammonium citrate (pH 5.4) at room temperature. Addition of 2 mM-adenine sulfate reduced twinning and "crystal shower". The resulting trigonal crystals diffract beyond 2.5 A resolution using a rotating anode X-ray generator. The space group was determined to be P3(1)21 or P3(2)21 (a = b = 103.9.A, c = 134.6 A, alpha = beta = 90 degrees, gamma = 120 degrees) based on their precession photography of h0l and hk0 zones. There seems to be three monomers in an asymmetric unit for VM = 2.51 A3/Da.
Insights
Mirabilis antiviral protein (MAP) crystallization was optimized for X-ray crystallography. High-quality trigonal crystals were obtained, enabling structural analysis of this ribosome-inactivating protein.
Area of Science:
- Biochemistry
- Structural Biology
- Crystallography
Background:
- Mirabilis antiviral protein (MAP) is a ribosome-inactivating protein from Mirabilis jalapa L.
- MAP exhibits antiviral properties, making its structural characterization important.
Purpose of the Study:
- To obtain high-quality crystals of MAP suitable for X-ray crystallography.
- To determine the crystal structure of MAP.
Main Methods:
- Optimization of crystallization conditions using the hanging drop vapor diffusion method.
- Crystal screening with varying ammonium sulfate and ammonium citrate concentrations.
- X-ray diffraction analysis of trigonal crystals.
Main Results:
- Crystallographic quality crystals of MAP were successfully grown.
- Trigonal crystals diffracted beyond 2.5 Å resolution.
- The space group was determined as P3(1)21 or P3(2)21, with three monomers per asymmetric unit.
Conclusions:
- Optimized crystallization protocols enable structural studies of MAP.
- The determined crystal parameters provide a basis for further structural elucidation of MAP's antiviral mechanism.