Journal of Molecular Biology
CommunicationThe Structure and Conformation of Lys63-Linked Tetraubiquitin
Section snippets
Experimental details
Lys63-linked tetra-Ub chain was synthesized using two Ub mutants (K48R–K63R and Ub-D77) following Pickart and Raasi.23 All the enzymes required for this synthesis, viz., E1 (human), Ubc13/MMS2 (yeast), and Yuh1 (yeast), were also expressed and purified as recombinant proteins in Escherichia coli. After the chain of required length was synthesized, it was subjected to a final step of purification using size-exclusion chromatography in Superdex 75 prep-grade 26/60 column. The protein was then
Accession number
Coordinates and structure factors have been deposited in the Protein Data Bank with accession number 3HM3.
Acknowledgements
This work was supported by the Howard Hughes Medical Institute (A.B.D and C.W.) and the National Cancer Institute (SBDR grant no. CA92584; G.H.). We thank Michal Hammel from the Lawrence Berkeley National Laboratory for helping with the implementation of MES.
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