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. 2015 Oct;71(Pt 10):1247-50.
doi: 10.1107/S2053230X15015447. Epub 2015 Sep 23.

Purification, crystallization and X-ray crystallographic analysis of human RAB11(S20V), a constitutively active GTP-binding form

Chang Min Kim  1 , Jae Young Choi  1 , Jong Hwan Yoon  1 , Hyun Ho Park  1
Affiliations

Purification, crystallization and X-ray crystallographic analysis of human RAB11(S20V), a constitutively active GTP-binding form

Chang Min Kim et al. Acta Crystallogr F Struct Biol Commun. 2015 Oct.

Abstract

RAB11, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking during endosome recycling. Substitution of Ser20 by Val20 in Rab11 [RAB11(S20V)] inhibits its GTP hydrolysis activity and produces a constitutively active GTP-binding form. In this study, the RAB11(S20V) mutant was overexpressed in Escherichia coli with an engineered C-terminal His tag. RAB11(S20V) was then purified to homogeneity and was crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.4 Å from a crystal belonging to space group I4, with unit-cell parameters a = 74.11, b = 74.11, c = 149.44 Å. The asymmetric unit was estimated to contain two molecules of RAB11(S20V).

Keywords: RAB11; crystallization; diffraction; membrane trafficking; small G protein.

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Figures

Figure 1
Figure 1
Purification of RAB11(S20V) by size-exclusion chromatography (SEC). The insert shows the SEC fractions analyzed by SDS–PAGE.
Figure 2
Figure 2
Crystals of RAB11(S20V). The crystals grew in 5 d in the presence of 2.4 M sodium chloride, 0.1 M magnesium chloride, 0.1 M Tris–HCl pH 7.1. The approximate dimensions of the crystals were 0.1 × 0.1 × 0.1 mm.
Figure 3
Figure 3
A diffraction image (1° oscillation) of the Rab11(S20V) crystal. The 2.5 Å resolution ring is indicated; the crystal data were processed to 2.4 Å resolution.
See this image and copyright information in PMC

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