Article info 2017 5 (2)    005  (02)   pp.  82 ~ 87
Title Purification, crystallization and X-ray crystallographic analysis of RabA1a from Arabidopsis thaliana
Authors Ji-Sook Yun and Jeong Ho Chang*
Institutions Department of Biology Education, Kyungpook National University, Daehak-ro 80, Buk-gu, Daegu 41566, South Korea. *Correspondence: jhcbio@knu.ac.kr
Abstract RabA1a is a small GTPase that contain a conserved GTP binding pocket to promote GTP hydrolysis. Fifty-seven isotypes of Rab GTPase have been found in Arabidopsis thaliana, and the twenty-seven RabA proteins are classified into six subgroups, from RabA1 to RabA6. RabA members play crucial roles in controlling docking and fusion during vesicle transport, yet their molecular and physiological functions remain unknown. Moreover, no RabA group protein structures have yet been determined. In this study, in order to understand the structural features and regulatory mechanisms of RabA from the crystal structures, an engineered N-terminal His-tagged RabA1a protein was overexpressed and purified. The RabA1a protein was crystallized with either GDP for an inactive form or GppNHp as a non-hydrolyzable GTP analog for an active form. X-ray diffraction data from crystals of RabA1a-GDP and RabA1a-GppNHp complexes were collected to resolutions of 2.8 Å and 2.6 Å, respectively. The crystal of the RabA1a-GDP complex belonged to the space group P2 1 , with unit cell parameters a = 36.7, b = 70.4, c = 68.0 Å, and β = 97.2°. The crystal of the RabA1a-GppNHp complex belonged to the space group P1, with unit cell parameters, a = 37.6, b = 48.4, c = 68.5 Å, α = 88.5, β = 74.1, and γ = 84.4°. Both crystals contained two macromolecules each containing GDP or GppNHp in the asymmetric units.