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100605 - BE.342/442 Thursday October 6 2005 Topic Practical...

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BE.342/442 Thursday, October 6, 2005 Topic: Practical Aspects of Single Crystal X-ray Crystallography, Part 2 X-ray diffraction was key to solving structures such as RNA and collagen. However, X-ray crystallography of fibers is extremely difficult, because the fibers entangle rather than aligning in a crystalline structure. Important developments in structural biology in the last decade include: expression of proteins via gene coding (rather than purification from tissue! one exception is rhodopsin, which is sometimes purified from cow’s eye), availability of beamlines at synchrotron radiation sources, and computing hardware and integrated software packages. How is synchrotron radiation produced? (E.g, CERN in Geneva, Switzerland.) Charged particles are bent around a ring, releasing high-energy light tangential to the curve of their path. The highest available energies have been climbing steadily since the 1960’s with X-ray tubes, until modern third-generation light sources such as ESRF, which have a billion times the brilliance of x-ray tubes. At the frontier of structural biology is the structural determination of the nucleosome core, where DNA is wrapped around histones (1997) 8 proteins 146 DNA base pairs 52 X-ray reflections the ribosome subunit 30S (2000) 21 proteins 1542 nucleotides 254 reflections the ribosome subunit 50S (2000) 35 proteins 3020 nucleotides 666,000 reflections and the bluetongue virus core (1998) 900 proteins 19,000 RNA bases 3,300,000 reflections Without synchrotron radiation, enormous structures such as the ribosome could not have been solved. Structural genomics aims to provide a structure or a model for every protein in all sequenced genomes. Enourmous funds have gone into structural genomics initiatives, especially from venture capitalist sources. However, the field has many challenges. For example, of the proteins
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