Technical Reference #1807

1807.

Disease mutations in Rab7 result in unregulated nucleotide exchange and inappropriate activation Brett A. McCray; Emmanuel Skordalakes; and J. Paul Taylor, St. Jude Children's Research Hospital, Human Molecular Genetics, 19(1807), (2010)
Link To Paper

Materials & Methods:
Determination of the structure of L129F Rab7: Full-length L129F Rab7 was produced and crystallized as described in Supplementary Material. Phases were calculated by MR using PHASER (53) as implemented in CCP4 suite of programs using the substrate-free structure of Rab7 (PDB ID 1VG8) as a search model. Maps calculated after one cycle of refinement by REFMAC followed by solvent flattening and five-fold multidomain non-crystallographic symmetry averaging using density modification (54) revealed clear fo–fc density for the nucleotide substrate at 2.5 s contour level. Model building was carried out in COOT (55) and the model was refined using both CNS-SOLVE (56) and REFMAC5 (57). The last cycles of refinement were carried out with translation libration screw (TLS) restraints as implemented in REFMAC5 and without NCS restraints (Table 1).

Microscopic Technique
Confocal Microscopy

Cell Type(s)
HEK293T cells; HeLa cells