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| Soichi Wakatsuki - | |
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Glycosylation is one of the most important post-translational modifications
carried out by an intricate network of glycosyltransferases, glycosidases,
transporters, and transport proteins in the endoplasmic reticulum (ER) and
the Golgi apparatus. It is strongly coupled to intracellular protein
transport between different organelles, where protein-protein interactions
play key roles. We are pursuing a target-oriented structural proteomics
project on protein glycosylation and transport. The structures of the
three domains of GGA, a new class of adapter proteins of vesicular transport
(Nature 415, 937-41, 2002; Nature Structural Biology, 9, 527-31, 2002; ibid.
10, 386-93, 2003, J.B.C., 279, 7105-11279, 2004, Traffic, 5, 437-448, 2004)
will be used to illustrate our approach. In addition, several other examples
of recent X-ray structures will be presented from plant lectins responsible
for trafficking of glycosylated proteins between the ER and the Golgi
apparatus, mammalian glycosyltransferases (J.B.C., 279, 22693-703, 2004)
and glycosidases.
In these studies, we often encounter difficulties in solving complex
structures of multi-domain proteins: protein production, domain boundaries,
crystallization, small crystals, large unit cells, etc. In an effort to
solve some of these problems, we are developing a number of high-throughput
technologies for structural genomics but also extremely demanding
crystallographic projects. We have installed two new insertion-device
MAD beam lines where a high quality data set can be collected in 5~30
minutes. They are equipped with user-friendly software and highly accurate
diffractometers (1~2 microns rotational error). The diffractometer will
be further improved to 100 nm rotational error for a new micro-focus beam
line at PF. A large scale crystallization robot (200,000 conditions/day)
and robotics for crystal harvesting and cryo-protectant exchange are being
developed. Two SSRL robot systems have been installed and are being
commissioned for rapid exchange of crystals in X-ray hutches, which will
accelerate data collection efficiency from about 20/day to more than
100/day for uninterrupted data collection of up to 288 crystals.
We are also developing a next generation X-ray area detector, X-ray HARP,
based on the avalanche phenomenon of amorphous selenium, for continuous
and super-fine phi slicing data collection from weakly diffracting
crystals. With high sensitivity (at least 100 times as compared to CCD
detectors) and fast readout (30 to 90 frames/sec, and this can be
extended to 120 frames/sec, a repetition rate of X-FEL, in the future),
the long term goal of this detector development is an ultra-sensitive
fast area detector for single particle structural analysis or nano-meter
scale protein crystallography using X-FEL or ERL.
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