Crystallization and Preliminary X-Ray Diffraction Analysis of PD-L4, a Ribosome Inactivating Protein from Phytolacca dioica L. leaves

Abstract

PD-L4, a type 1 ribosome inactivating protein from Phytolacca dioica leaves, has been successfully crystallized using vapour diffusion methods and PEG 4000 as a precipitant agent. In addition, crystals of a PD-L4 mutant, which has been recently observed to have a lower polynucleotide-adenosine glycosidase activity on DNA, rRNA and poly (A) substrates, have been obtained. To gather information on PD-L4 reaction mechanism both forms have been co-crystallized with adenine, the major product of their catalytic reaction. Diffraction patterns extend to atomic resolution and crystals belong to the orthorhombic P2(1)2(1)2(1) space group, with one molecule in the asymmetric unit. Structure determination has been achieved using molecular replacement; preliminary electron density maps have clearly given evidence of adenine binding.

Crystallization and Preliminary X-Ray Diffraction Analysis of PD-L1, a Highly Glycosylated Ribosome Inactivating Protein with DNase Activity

“…Matthews coefficient calculations (Matthews, 1968) Molecular replacement using the program "Il Milione" [25] and the structure of the ribosome inactivating protein from Phytolacca americana (PDB code 1qci) as a starting model [26] resulted in a clear solution, with two molecules in the asymmetric unit and a correlation factor of 0.38. Similar results were obtained using the structure of PD-L4 from Phytolacca dioica as a starting model [30]. Manual and automatic model-building sessions, aimed at defining both the structures of the protein and of the glycan chains, are in progress using the programs ARP/wARP [31] and O [28].…”

Atomic resolution (1.1 Å) structure of the ribosome‐inactivating protein PD‐L4 from Phytolacca dioica L. leaves

“…Crystallization trials were conducted using the hanging‐drop vapor diffusion technique as previously reported 30. Best crystals of wt PD‐L4, of size 0.1 mm × 0.1 mm × 0.4 mm, were obtained in about 4 days using a protein concentration of 8 mg mL −1 in the presence of 20% (w/v) PEG 4000, 10% (v/v) isopropanol, and 0.1 M Hepes (pH 7.5).…”