What is available for structural analysis?
Biomolecular nuclear magnetic resonance spectroscopy (NMR) is a very versatile technique that can be used to determine the three dimensional structures of proteins, DNA, RNA, oligosaccharides and small molecules. It can also be used to study conformational changes, intermolecular interactions (both strong and weak), and molecular motion on many timescales in solution. NMR is widely used in metabolomics studies. (and enquiries for this type of application should be directed to Glasgow Polyomics) Solution state NMR complements the other techniques, such as circular dichroism and X-ray crystallography, available in the facility.
X-Ray Crystallography can be used to determine the 3D structure of proteins. The process begins with trying to produce well-ordered single crystals of the protein of interest that are large enough to diffract X-rays to high resolution and then to be able to go on and solve their 3D structure. Here in Glasgow with the use of a variety of commercial sparse matrix screens, coupled with the use of crystallisation robots, we can efficiently test for initial crystallisations conditions that can be optimised to produce diffracting quality crystals, which can be assessed in-house in our X-ray diffractometer.
NMR Sample Criteria
For biomacromolecules (proteins, DNA and RNA), samples should typically be >95% pure in low (up to 100 mM) ionic strength, aqueous buffer that contains only hydrogen atoms that do exchange with the solvent, e.g. sodium phosphate.
The sample should be stable in solution for hours to weeks at the required temperature (typically 15-40 °C)
The buffer used should not contain additives such as glycerol, detergents and reducing agents. Alternatively, deuterated buffer components (e.g. Tris, HEPES, imidazole, EDTA, DTT, TCEP) should be used.
Biomacromolecule concentrations should ideally be in the range 0.1-2.0 mM. The standard NMR sample volume is 600 ul meaning that 60 nmoles to 1.2 umoles of the target will be required per sample.
Contact Dr Brian Smith
X-Ray Crystallography Sample Criteria
120ul of protein concentration approx. 10mg/ml for each crystal screen tray. There are many commercial crystallisation screens available
https://www.moleculardimensions.com/products/c255-Crystallization-Screens but an initial selection would include:
For membrane proteins; Memplus, memsys, memgold
For soluble proteins; PACT, MIDAS, JSCG, Morpheus+
Cost: £50 for each tray. Thereafter discussions to cost future projects
Contact Professor Helen Walden