Structure determination of biological macromolecules by X-ray diffraction



Structure determination of biological macromolecules by X-ray diffraction


Rocco Caliandro
Dritan Siliqi
Sabino Maggi
Sara Depinto* Postdoc fellow
Benny Danilo Belviso* PhD fellow


Commessa PM.P07.011: Development and application of computational methods for biomolecule structure determination
Modulo I: Development and application of computational methods for structure determination by X-ray diffraction


Crystallography, protein crystallization, X-ray diffraction

G. Natile and F. Arnesano, University of Bari, Italy.
M. Trotta, Institute for chemical and physical processes, IPCF, CNR, Bari, Italy.
G. Diprofio, Institute for Membrane Technology, ITM, CNR, Cosenza, Italy.
E. Curcio and E: Drioli, Department of Chemical and Materials Engineering, University of Calabria, Cosenza, Italy.
L. Penarrubia, University of Valencia, Spain.
G. Falini, University of Bologna, Italy.

Experimental activities of protein crystal structure determination are carried out. They include the following steps:
a) protein purification via chromatography;
b) protein crystallization by hanging drop and sitting drop techniques;
c) data taking at synchrotron sources;
d) data analysis through the crystallographic software packages IL MILIONE and PHENIX to perform initial phasing, phase extension and refinement, model building, completion and refinement;
e) model validation and deposition in the Protein Structural Database.
Current investigations include:
- complexes of human ubiquitin with group-12 metal ions;
- adducts of hyman ubiquitin with Pt-based drugs (cisplatin, Zeise's salt);
- complexes of horse heart cytochrome with chromate.
- Structure determination of the Arabidopsis thaliana copper chaperone

Experimental phasing activities are carried out. A Br-based compound is added to crystallization solutions. Protein crystals are formed, which include these compounds. Ad-hoc crystallographic methods based on Patterson deconvolution are developed and applied to diffraction data analysis to improve the crystal structure solution of the protein driven by the specific Br-Br patterns of the included compound.
New protein crystallization methods are attempted, based on the use of microporous hydrophobic membranes. They allow a greater control of the crystal growth process and allow to obtain protein crystals when conventional vapour-diffusion methods fails.
Experiments of X-ray spectroscopy on metallo-proteins in the near-edge (XANES) and extended (EXAFS) regions of the X-ray absorption spectrum. The aim is study changes in the oxidation state of the heavy-atom involved and to reconstruct its first coordination shell. Current investigations include Pt binding sites in Atox1 and measure of Cr(IV) reduction rate to Cr(III) by reaction with cytochrome.


Structure determination of biological macromolecules by X-ray diffraction - Img

Crystal structures of Pt(II) and Zn(II) complexes of human ubiquitin, (reported in red and yellow, respectively) compared with previous results on adducts of human ubiquitin with group-12 metal ions (reported in blue).


Structure determination of biological macromolecules by X-ray diffraction - IMG

Crystal packing of the Zn3-hUb adduct. Only the network formed by chain A is shown. The second molecule in the asymmetric unit (chain B) gives a similar network (not shown) which fills the empty spaces left from the former network. The Zn(II) ions are shown as spheres and those of symmetry-related hUb molecules are labelled with (*), (**), and (***).


Rocco Caliandro
Email :
Tel. : +39/080/5929150

Last Updated (Thursday, 02 December 2010 16:06)