Abstract
High-throughput crystallography requires a method by which the structures of proteins can be determined quickly and easily. Experimental phasing is an essential technique in determining the three-dimensional protein structures using single-crystal X-ray diffraction. In macromolecular crystallography, the phases are derived either by Molecular Replacement (MR) method using the atomic coordinates of a structurally similar protein or by locating the positions of heavy atoms that are intrinsic to the protein or that have been added (MIR, MIRAS, SIR, SIRAS, MAD and SAD). Availability of in-house lab data collection sources (Cu Kα and Cr Kα radiation), cryo-crystallography and improved software for heavy atom location and density modification have increased the ability to solve protein structures using SAD. SAD phasing using intrinsic anomalous scatterers like sulfur, chlorine, calcium, manganese and zinc, which are already present in the protein becomes increasingly attractive owing to the advanced phasing methods. An analysis of successful SAD phasing on three proteins, lysozyme, glucose isomerase and thermolysin based on the signal of weak anomalous scatterers such as sulfur atom and chloride ion have been carried out. This analysis also proves that even the anomalous signal provided or present naturally in a macromolecule is good enough to solve crystal structures successfully using lab source chromium-generated X-ray radiation.
Highlights
The sequencing of complete genomes has spurred a growing need for structural information on the encoded gene products that has driven the development of highthroughput crystallography [1,2,3,4,5,6,7]
High-throughput crystallography requires a method by which the structures of proteins can be determined quickly and
Single-wavelength Anomalous Diffraction (SAD) phasing using intrinsic anomalous scatterers like sulfur, chlorine, calcium, manganese and zinc, which are already present in the protein becomes increasingly attractive owing to the advanced phasing methods
Summary
The sequencing of complete genomes has spurred a growing need for structural information on the encoded gene products that has driven the development of highthroughput crystallography [1,2,3,4,5,6,7]. The power of Single-wavelength Anomalous Diffraction (SAD) was illustrated even 25 years ago and the anomalous signal from six sulfur atoms was used to solve the structure of crambin, a 4.8 kDa protein [9,10]. SAD has become the choice for phasing [11,12,13] after gaining advantage over the Multi-wavelength Anomalous Diffraction, MAD [14]. Availability of synchrotron radiation, cryo-crystallography and improved software for heavy atom location and density modification have increased the ability to solve protein structures using SAD. In the absence of synchrotron radiation, SAD method using rotating anode generator (Cu Kα and Cr Kα) lab source data is mostly used to solve the phase problem in X-ray crystallography. Sufficiently accurate anomalous data can be collected in the laboratory using Cu Kα and Cr Kα radiations [16,17,18,19,20]
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