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Figure 1.
Figure 1. (a) Verification of the ubiquitin species contained
within our crystals. Lane 1, purified tri-ubiquitin; Lane 2,
purified di-ubiquitin; Lanes 3 and 4, molecular weight markers
(molecular weights are shown in the space between the two gels);
Lane 5, a different sample of purified di-ubiquitin that was
used for crystallization experiments; Lane 6, washed and
dissolved di-ubiquitin crystals; Lane 7, washed and dissolved
tri-ubiquitin crystals. Crystals were washed repeatedly with
protein-free mother liquor, transferred to sample buffer and run
on a 12-20% SDS-PAGE gradient gel, which was fixed and stained
with Coomassie Brilliant Blue. The formation of SDS-resistant
higher molecular weight species is likely due to residual PEG in
the dissolved crystals. (b) Structure of the di-ubiquitin chain
found in the asymmetric unit of both the di- and tri-ubiquitin
crystals. The distal molecule is colored cyan and the proximal
molecule yellow. The side chains of Lys-63 (on the proximal
molecule) and Arg-63 (on the distal molecule) are shown in
ball-and-stick representation. (c) Positional disorder in the
tri-ubiquitin structure. At top is shown a portion of one of the
extended ubiquitin chains running throughout the crystal; the
distal and proximal ends of the chain are marked. Three adjacent
asymmetric units are shown (A  -B
,
A-B, and A -B
),
separated by dotted lines. Below is a schematic representation
of the packing of the tri-ubiquitin species. The distal-most
subunit of the trimer will alternately occupy the A and B
positions in the asymmetric unit. Figures 1 and 2 were prepared
using MacPyMol (http://www.pymol.org).
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