Introduction to phasing
- Centre for Biomolecular Sciences, University of St Andrews, St Andrews, Fife KY16 9ST, Scotland (United Kingdom)
This introductory paper to the CCP4 weekend on experimental phasing introduces the concept of the ‘phase problem’ for non-experts. Modern methods of phasing are explored, including some recent examples that can be downloaded as tutorials. When collecting X-ray diffraction data from a crystal, we measure the intensities of the diffracted waves scattered from a series of planes that we can imagine slicing through the crystal in all directions. From these intensities we derive the amplitudes of the scattered waves, but in the experiment we lose the phase information; that is, how we offset these waves when we add them together to reconstruct an image of our molecule. This is generally known as the ‘phase problem’. We can only derive the phases from some knowledge of the molecular structure. In small-molecule crystallography, some basic assumptions about atomicity give rise to relationships between the amplitudes from which phase information can be extracted. In protein crystallography, these ab initio methods can only be used in the rare cases in which there are data to at least 1.2 Å resolution. For the majority of cases in protein crystallography phases are derived either by using the atomic coordinates of a structurally similar protein (molecular replacement) or by finding the positions of heavy atoms that are intrinsic to the protein or that have been added (methods such as MIR, MIRAS, SIR, SIRAS, MAD, SAD or combinations of these). The pioneering work of Perutz, Kendrew, Blow, Crick and others developed the methods of isomorphous replacement: adding electron-dense atoms to the protein without disturbing the protein structure. Nowadays, methods from small-molecule crystallography can be used to find the heavy-atom substructure and the phases for the whole protein can be bootstrapped from this prior knowledge. More recently, improved X-ray sources, detectors and software have led to the routine use of anomalous scattering to obtain phase information from either incorporated selenium or intrinsic sulfurs. In the best cases, only a single set of X-ray data (SAD) is required to provide the positions of the anomalous scatters, which together with density-modification procedures can reveal the structure of the complete protein.
- OSTI ID:
- 22347931
- Journal Information:
- Acta Crystallographica. Section D: Biological Crystallography, Vol. 66, Issue Pt 4; Other Information: PMCID: PMC2852296; PMID: 20382985; PUBLISHER-ID: ba5147; OAI: oai:pubmedcentral.nih.gov:2852296; Copyright (c) Taylor 2010; This is an open-access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original authors and source are cited.; Country of input: International Atomic Energy Agency (IAEA); ISSN 0907-4449
- Country of Publication:
- Denmark
- Language:
- English
Similar Records
Anomalous Diffraction at Ultra-High Energy for Protein Crystallography
Direct phase selection of initial phases from single-wavelength anomalous dispersion (SAD) for the improvement of electron density and ab initio structure determination