While the crystallographic phase problem is definitively solved in
practice far small molecules, it is still a challenging problem in
Macromolecular Crystallography. This is one of the most exciting areas
of the modern Sciences and constitutes a primary tool for studying the
structure of complex biological systems, so providing insight into the
mechanisms at the basis of the life. Indeed Macromolecular
Crystallography allows to determine the crystal structure of proteins,
nucleic acids, viruses, etc. and provides detailed understanding of a
large variety of important living processes such as photosynthesis,
transmission of hereditary information, viral infections, etc.
Of paramount importance far Macromolecular Crystallography are the
Crystallographic Methods for the solution of crystal structures: their
efficacy is responsible for the success or the failure of the research,
and governs the economical aspects of the activity (innovation in the
methods can speed up the research, allows to spare man-time and
resources, improves the quality of the results, facilitates the creation
of Companies far Research and Scientific Services).
Usually the molecular complexity and the low data resolution make native
protein diffraction data insufficient far the crystal structure solution.
Additional (and expensive) efforts are then necessary to prepare
isomorphous derivatives and collect additional data, and/or to use
anomalous dispersion effects to increase the experimental information
available far the solution of the phase problem. Ab initio techniques,
SIR (Single Isomorphous Replacement), MIR (Multiple Isomorphous
Replacement), SAS (Single Anomalous Scattering), MIRAS (the combination
of MIR with Anomalous Scattering) MAD (Multiple Anomalous Dispersion),
and MR ( Molecular Replacement) are the current techniques used to solve
a completely unknown macromolecular structure.
Presentation of the research
The research developed by the Istituto di Cristallografia has brought
quite relevant contributions to most of the above fields. In particular:
a)ab initio techniques have been developed which move the limit of
the data resolution from atomic to 1.4Angstrom, so dramatically
extending the set of structures approachable by ab initio techniques;
b) the SIR-MIR-SIRAS-MIRAS-SAD-MAD techniques have been integrated
with a new Direct Methods approach: this has been enabled to englobe
the errors (from the experiment, or from the model or from the lack of
isomorphism, or ...) into the set of primitive random variables of the
probabilistic approach. The method of the joint probability distribution
function has been the mathematical tool used for the derivation of the
new formulas.
The new probabilistic theories have been implemented into packages for
the automatic solution of the phase problem in macromolecular
crystallography. The applications to several real cases show the high
efficiency of the new approach, its robustness against the experimental
errors, its ability in providing automatically a sensible structural
model of the protein, practically without any user intervention. The set
of programs constitute a unvaluable tool for the development of the
high-throughput structural genomics, and will be soon made available to
all the scientific community.
The research is at the moment financied by the CNR annual contribution.
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