X-ray crystallography for macromolecular complexes
Organismal bodies and biogenic activities are sustained by various macromolecules including proteins, DNA, and RNA. These bio-molecules express their functions or activities by interacting with each other or by forming gigantic molecular complexes. Studying the three-dimensional structures of these bio-molecules allows us to understand their action mechanism. We employ X-ray crystallography for the structural analyses. The obtained three-dimensional structures provide not only insights into detailed molecular mechanisms, but also firm bases for applied researches such as drug development.
- Preparation and reconstitution of molecular complexes
Macromolecular complexes of interest are prepared by various means. For example, the constituents of the complexes, such as proteins and nucleic acids, are separately prepared in high purity, and they are assembled in vitro. Alternatively, the components are co-expressed in cells, from which the complex is isolated. Endogenous protein complexes are also purified from cells by means of affinity tags.
- Crystallization of the complexes
The complexes are crystallized for structure analyses. To stabilize the conformations and interactions within the complexes, inhibitors and/or intra- and inter-molecular crosslinks are employed. Crystallization robots allow screening thousands of crystallization conditions while consuming minimal quantity of the sample.
- Data acquisition and structural analyses
X-ray diffraction data are collected from the obtained crystals. Synchrotron radiation is often used for the data collection. By using the obtained X-ray diffraction data, structure determination is achieved.