Crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5

Abstract

Death-associated protein 5 (DAP5) is a member of the eIF4G family of scaffolding proteins that mediate cap-independent translation initiation by recruiting the translational machinery to internal ribosomal entry sites (IRESs) on mRNA. The MIF4G domain of DAP5 directly interacts with the eukaryotic initiation factors eIF4A and eIF3 and enhances the translation of several viral and cellular IRESs. Here, the crystallization and preliminary X-ray diffraction analysis of the MIF4G domain of DAP5 is presented.

Figure 1

Analytical size-exclusion chromatography profile and SDS–PAGE analysis (inset) of purified DAP5M. Size-exclusion chromatography was carried out on a Superdex 75 column (GE Healthcare, Little Chalfont, England). SDS–PAGE was carried out on a 12% polyacrylamide gel and the protein was visualized by Coomassie Brilliant Blue staining. Numbers indicate the migration of protein molecular-weight markers in kDa.

Figure 2

Mass-spectrometric analysis of purified DAP5M: (a) native, (b) selenomethionine labelled.

Figure 3

DAP5M crystal forms A (a) and B (b).

Figure 4

Diffraction patterns of crystal forms A (a) and B (b) collected in-house on a Rigaku MicroMax-007 HF microfocus X-ray generator fitted with Varimax X-ray optics and a Saturn 944+ CCD detector. Resolution rings are shown in red. Resolutions are given in Å.

Figure 5

The κ = 180° section of the self-rotation function for DAP5M data using data in the 12–3 Å resolution range and a Patterson cutoff radius of 21 Å. The crystallographic twofold is found at the centre of the stereogram, whereas the twofold NCS axis is at ϕ = 64°, ψ = 65°, κ = 180°.