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On the influence of crystal size and wavelength on native SAD phasing
- Source :
- Acta Crystallographica Section D Structural Biology. 72:728-741
- Publication Year :
- 2016
- Publisher :
- International Union of Crystallography (IUCr), 2016.
-
Abstract
- Native SAD is an emerging phasing technique that uses the anomalous signal of native heavy atoms to obtain crystallographic phases. The method does not require specific sample preparation to add anomalous scatterers, as the light atoms contained in the native sample are used as marker atoms. The most abundant anomalous scatterer used for native SAD, which is present in almost all proteins, is sulfur. However, the absorption edge of sulfur is at low energy (2.472 keV = 5.016 Å), which makes it challenging to carry out native SAD phasing experiments as most synchrotron beamlines are optimized for shorter wavelength ranges where the anomalous signal of sulfur is weak; for longer wavelengths, which produce larger anomalous differences, the absorption of X-rays by the sample, solvent, loop and surrounding medium (e.g.air) increases tremendously. Therefore, a compromise has to be found between measuring strong anomalous signal and minimizing absorption. It was thus hypothesized that shorter wavelengths should be used for large crystals and longer wavelengths for small crystals, but no thorough experimental analyses have been reported to date. To study the influence of crystal size and wavelength, native SAD experiments were carried out at different wavelengths (1.9 and 2.7 Å with a helium cone; 3.0 and 3.3 Å with a helium chamber) using lysozyme and ferredoxin reductase crystals of various sizes. For the tested crystals, the results suggest that larger sample sizes do not have a detrimental effect on native SAD data and that long wavelengths give a clear advantage with small samples compared with short wavelengths. The resolution dependency of substructure determination was analyzed and showed that high-symmetry crystals with small unit cells require higher resolution for the successful placement of heavy atoms.
- Subjects :
- 0301 basic medicine
Materials science
Protein Conformation
chemistry.chemical_element
02 engineering and technology
Crystallography, X-Ray
Molecular physics
law.invention
Crystal
03 medical and health sciences
Bacterial Proteins
Structural Biology
law
Pseudomonas
Animals
Absorption (electromagnetic radiation)
Helium
Quantitative Biology::Biomolecules
X-Rays
Resolution (electron density)
Proteins
Equipment Design
021001 nanoscience & nanotechnology
Synchrotron
Wavelength
Crystallography
030104 developmental biology
chemistry
Absorption edge
Ferredoxins
Substructure
Muramidase
Crystallization
Oxidoreductases
0210 nano-technology
Chickens
Sulfur
Subjects
Details
- ISSN :
- 20597983
- Volume :
- 72
- Database :
- OpenAIRE
- Journal :
- Acta Crystallographica Section D Structural Biology
- Accession number :
- edsair.doi.dedup.....eae2cd4021113f144b17f0eb42b7dfef
- Full Text :
- https://doi.org/10.1107/s2059798316005349