Double-Lanthanide-Binding Tags for Macromolecular Crystallographic Structure Determination
A double-lanthanide-binding tag (dLBT), a small peptide sequence engineered to bind two lanthanide ions (e.g., Tb{sup 3+}) with high affinity, was used to solve the phase problem for the structure determination of ubiquitin by the single-wavelength anomalous diffraction (SAD) method. Since the dLBT is comprised exclusively of encoded amino acids, the necessity for the incorporation of unnatural amino acids or chemical modification of the protein as a prerequisite for X-ray structure determination is eliminated. A construct encoding the dLBT as an N-terminal fusion with ubiquitin provides for facile expression and purification using standard methods. Phasing of the single-wavelength X-ray data (at 2.6 {angstrom} resolution) using only the anomalous signal from the two tightly bound Tb{sup 3+} ions in the dLBT led to clear electron-density maps. Nearly 75% of the ubiquitin structure was built using automated model-building software without user intervention. It is anticipated that this technique will be broadly applicable, complementing existing macromolecular phasing methodologies. The dLBT should be particularly useful in cases where protein derivatization with heavy atoms proves to be problematic or synchrotron facilities are unavailable.
- Research Organization:
- Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
- Sponsoring Organization:
- Doe - Office Of Science
- DOE Contract Number:
- DE-AC02-98CH10886
- OSTI ID:
- 930450
- Report Number(s):
- BNL-81202-2008-JA; JACSAT; TRN: US200904%%722
- Journal Information:
- Journal of the American Chemical Society, Vol. 129; ISSN 0002-7863
- Country of Publication:
- United States
- Language:
- English
Similar Records
Multi-crystal Anomalous Diffraction for Low-resolution Macromolecular Phasing
Lanthanide-Binding Tags for 3D X-ray Imaging of Proteins in Cells at Nanoscale Resolution