Principles of X-ray crystallography
Scattering
- When light hits matter…
- Vibration\(\rightarrow\)scattering (all directions)
- Energy level transitions\(\rightarrow\)absorption and emission (fluorescence)
- Photochemical reactions (e.g. photosynthesis)
- scattering can give rise to refraction and diffraction
- experiments on scattering
- turbidity (reduction in intensity)
- angular dependence
- changes in \(\lambda\)
Wnt3-Fz8 Complex (Hirai et al. 2019)
- crystals of lysine-methylated and deglycosylated human Wnt3 (hWnt3)-mFz8 CRD complex were obtained by X-ray crystal structure was solved by molecular replacement and refined to a resolution of 2.8Å.
Difficulties in crystallisation and their solutions
- Strong hydrophobic property of Wnt proteins caused by a covalent lipid modification
Optimisation and chemical modifications conducted to ensure high expression yields, enhanced solubility and sample homogeneity
Solubility
- Failed attempts on making crystallization constructs
- afamin can solubilize Wnt proteins; when coexpressed and complexed, Wnt3 and 3a can be purified to homogeneity. However, diffraction-quality crystals could not be obtained after repeated trials.
- coexpression with mFz8 CRD after Janda’s success; but found purified Wnt3a-Fz8 CRD complex still remained hydrophobic and required detergents during concentration, which hampered crystallisation
- Successful: N-terminal truncation of Wnts to mimic the cleavage of the N-terminal peptide by a metalloprotease Tiki, which has been reported to reduce the overall hydrophobicity
- N-terminal 20 residues were removed from hWnt3/mWnt3a constructs
- PA-hWnt3(\(\Delta\)N)/PA-mWnt3a(\(\Delta\)N) coexpressed with mFz8 CRD C-terminally fused with modified human Fc.
- confirmed that complexes were fully soluble in aqueous buffer and could be concentrated to > 5 mg/ml without detergents
Optimisation
- Initially (following Janda) attached normal (with hinge region) human IgG1 Fc to the C-terminal of mFz8 CRD, intervened by a TEV protease cleavage sequence for the later Fc removal
- although complex formed with high yield, it could not be cleaved at all; different linker lengths showed no improvements
- decided to use IdeS protease to remove Fc.
Hirai, Hidenori, Kyoko Matoba, Emiko Mihara, Takao Arimori, and Junichi Takagi. 2019. “Crystal Structure of a Mammalian Wnt–Frizzled Complex.” Nature Structural & Molecular Biology 26 (5): 372–79. https://doi.org/10.1038/s41594-019-0216-z.