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. 2014 Jan;70(Pt 1):123-6.
doi: 10.1107/S2053230X13033712. Epub 2013 Dec 24.

Purification, crystallization and preliminary X-ray analysis of the inverse F-BAR domain of the human srGAP2 protein

Hongpeng Wang  1 , Yan Zhang  1 , Zhenyi Zhang  1 , Wei Lin Jin  2 , Geng Wu  1
Affiliations

Purification, crystallization and preliminary X-ray analysis of the inverse F-BAR domain of the human srGAP2 protein

Hongpeng Wang et al. Acta Crystallogr F Struct Biol Commun. 2014 Jan.

Abstract

Bin-Amphiphysin-Rvs (BAR) domain proteins play essential roles in diverse cellular processes by inducing membrane invaginations or membrane protrusions. Among the BAR superfamily, the `classical' BAR and Fes/CIP4 homology BAR (F-BAR) subfamilies of proteins usually promote membrane invaginations, whereas the inverse BAR (I-BAR) subfamily generally incur membrane protrusions. Despite possessing an N-terminal F-BAR domain, the srGAP2 protein regulates neurite outgrowth and neuronal migration by causing membrane protrusions reminiscent of the activity of I-BAR domain proteins. In this study, the inverse F-BAR (IF-BAR) domain of human srGAP2 was overexpressed, purified and crystallized. The crystals of the srGAP2 IF-BAR domain protein diffracted to 3.50 Å resolution and belonged to space group P2(1). These results will facilitate further structural determination of the srGAP2 IF-BAR domain and the ultimate elucidation of its peculiar behaviour of inducing membrane protrusions rather than membrane invaginations.

Keywords: human srGAP2; inverse F-BAR domain.

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Figures

Figure 1
Figure 1
Gel-filtration chromatography profiles for the human srGAP2 IF-BAR domain protein. (a) Gel-filtration chromatography profile for human srGAP2 (residues 1–488) protein. Elution volumes of the standard molecular-weight marker are indicated above the chromatogram. (b) Gel-filtration chromatography profile for human srGAP2 (residues 15–347) protein.
Figure 2
Figure 2
The final purified IF-BAR domain of human srGAP2 protein (residues 15–347). Protein samples were analyzed using 12% SDS–PAGE followed by Coomassie Blue staining. Lane 1, protein molecular-mass marker (labelled in kDa). Lane 2, the final purified protein after the gel-filtration chromatography step. The arrow indicates the band corresponding to the F-BAR domain of human srGAP2 protein (residues 15–347).
Figure 3
Figure 3
A single crystal of the IF-BAR domain of human srGAP2 protein (residues 15–347). The maximum dimensions of the crystals are 0.05 × 0.05 × 0.45 mm.
Figure 4
Figure 4
A typical X-ray diffraction image of the IF-BAR domain of human srGAP2 protein (residues 15–347). The crystal diffraction data set was processed to 3.50 Å resolution using the HKL-2000 software (Otwinowski & Minor, 1997 ▶).
Figure 5
Figure 5
A stereographic projection of the κ = 180° section of the calculated self-rotation function of the processed diffraction data.
See this image and copyright information in PMC

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