1.) X-ray diffraction analysis and structure determination of proteins (MX)

X-ray diffraction analysis for determination of the three-dimensional structure of proteins at an atomic level is an essential method within structural biology. Numerous issues within fundamental science as well as in the context of special applications can be answered with a protein structure.

Examples are:

  • solving of catalytic mechanisms,
  • identification of amino acids suited for specific mutagenesis (protein engineering),
  • visualisation of protein-ligand-interactions, e. g. for determination or development of appropriate inhibitors/activators,
  • insights in structure-properties relationships for proteins or protein mutants involved in disease patterns.

To determine the three-dimensional structure of a protein by means of x-ray diffraction analysis the first and major crucial step is the generation of a highly ordered single crystal of the corresponding protein. This prerequisite marks the starting point of a labour-intensive, sometimes tedious, empirical process: the search for a suited crystallization condition. Having overcome that hurdle several complex steps follow in order to verify and optimize these first crystals before they are applicable for data collection.

The CSS as research- and service center provides all these essential working steps as full-service for all members of the HHU as well as for external users: starting with the search for initial crystallization conditions, followed by crystal optimization, data collection at synchrotrons and structure determination and refinement.

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2.) Small-Angle-X-ray-Scattering (SAXS)

With Small-Angle-X-ray-Scattering the size, shape and oligomeric state of biological macromolecules in solution can be determined. SAXS can provide detailed information within a scale of one to several hundred nanometers which represents the relevant range for macromolecular samples. Small-Angle-X-ray-Scattering allows to examine all samples in native aqueous solution without any marker or crystallization which can be of crucial advantage. A special possibility is the analysis of protein complexes and their stoichiometry as well as the spatial orientation of the individual subunits to each other.

For biological macromolecules (e.g. proteins, DNA, sugars, amphiphiles) SAXS can resolve the structure in solution which also includes transition state structures like protein complexes or -aggregates, dynamic processes like ligand binding, conformational changes and reaction cycles, as well as interactions between proteins. Additionally flexible parts within a protein can be identified (e. g. intrinsically disordered proteins).

The CSS has regular access for SAXS beam time at the synchrotrons in Hamburg and Grenoble and offers the data collection as service for all members of the HHU and external users.

Futhermore, the CSS is about to schedule in house SAXS experiments. This will allow to optimize macromolecular experiments performed at synchrotrons on one hand, on the other hand SAXS experiments will become accessible for small (anorganic or organic) molecules, colloids or cross-linked microgels.


3.) Buffer screening (proteins)

Finding an appropriate buffer in which a protein is stable for a certain time can be tricky. The CSS can support you by offering a buffer screen which covers all usual buffer systems from pH 4 to pH 9.5 in 0.5 or 1.0 steps. Combinations of these buffers with salts or others agents are also possible. In total we provide three screens with 96 individual buffers or mixes each. The protein concentration should have 0.25 mg/ml at minimum, for testing 96 conditions 15 µl of the protein solution are required.

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