Robust structural analysis of native biological macromolecules from multi-crystal anomalous diffraction data
- Brookhaven National Laboratory, Upton, NY 11973 (United States)
- Virginia Commonwealth University, Richmond, VA 23298 (United States)
Anomalous diffraction signals from typical native macromolecules are very weak, frustrating their use in structure determination. Here, native SAD procedures are described for enhancing the signal to noise in anomalous diffraction by using multiple crystals are described. Five applications demonstrate that truly routine structure determination is possible without the need for heavy atoms. Structure determinations for biological macromolecules that have no known structural antecedents typically involve the incorporation of heavier atoms than those found natively in biological molecules. Currently, selenomethionyl proteins analyzed using single- or multi-wavelength anomalous diffraction (SAD or MAD) data predominate for such de novo analyses. Naturally occurring metal ions such as zinc or iron often suffice in MAD or SAD experiments, and sulfur SAD has been an option since it was first demonstrated using crambin 30 years ago; however, SAD analyses of structures containing only light atoms (Z{sub max} ≤ 20) have not been common. Here, robust procedures for enhancing the signal to noise in measurements of anomalous diffraction by combining data collected from several crystals at a lower than usual X-ray energy are described. This multi-crystal native SAD method was applied in five structure determinations, using between five and 13 crystals to determine substructures of between four and 52 anomalous scatterers (Z ≤ 20) and then the full structures ranging from 127 to 1200 ordered residues per asymmetric unit at resolutions from 2.3 to 2.8 Å. Tests were devised to assure that all of the crystals used were statistically equivalent. Elemental identities for Ca, Cl, S, P and Mg were proven by f′′ scattering-factor refinements. The procedures are robust, indicating that truly routine structure determination of typical native macromolecules is realised. Synchrotron beamlines that are optimized for low-energy X-ray diffraction measurements will facilitate such direct structural analysis.
- OSTI ID:
- 22347857
- Journal Information:
- Acta Crystallographica. Section D: Biological Crystallography, Vol. 69, Issue Pt 7; Other Information: PMCID: PMC3689535; PMID: 23793158; PUBLISHER-ID: ba5189; OAI: oai:pubmedcentral.nih.gov:3689535; Copyright (c) Liu et al. 2013; 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
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