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We tested the general applicability of in situ proteolysis to form protein crystals suitable for structure determination by adding a protease (chymotrypsin or trypsin) digestion step to crystallization trials of 55 bacterial and 14 human proteins that had proven recalcitrant to our best efforts at crystallization or structure determination. This is a work(More)
NleG homologues constitute the largest family of Type 3 effectors delivered by pathogenic E. coli, with fourteen members in the enterohaemorrhagic (EHEC) O157:H7 strain alone. Identified recently as part of the non-LEE-encoded (Nle) effector set, this family remained uncharacterised and shared no sequence homology to other proteins including those of known(More)
The second round of the community-wide initiative Critical Assessment of automated Structure Determination of Proteins by NMR (CASD-NMR-2013) comprised ten blind target datasets, consisting of unprocessed spectral data, assigned chemical shift lists and unassigned NOESY peak and RDC lists, that were made available in both curated (i.e. manually refined) or(More)
In selecting a method to produce a recombinant protein, a researcher is faced with a bewildering array of choices as to where to start. To facilitate decision-making, we describe a consensus 'what to try first' strategy based on our collective analysis of the expression and purification of over 10,000 different proteins. This review presents methods that(More)
High-throughput structural proteomics is expected to generate considerable amounts of data on the progress of structure determination for many proteins. For each protein this includes information about cloning, expression, purification, biophysical characterization and structure determination via NMR spectroscopy or X-ray crystallography. It will be(More)
Only about half of non-membrane-bound proteins encoded by either bacterial or archaeal genomes are soluble when expressed in Escherichia coli (Yee et al., Proc Natl Acad Sci USA 2002;99:1825-1830; Christendat et al., Prog Biophys Mol Biol 200;73:339-345). This property limits genome-scale functional and structural proteomics studies, which depend on having(More)
A standardized protocol enabling rapid NMR data collection for high-quality protein structure determination is presented that allows one to capitalize on high spectrometer sensitivity: a set of five G-matrix Fourier transform NMR experiments for resonance assignment based on highly resolved 4D and 5D spectral information is acquired in conjunction with a(More)
With the near completion of many genome sequencing projects has come the sobering realisation that our understanding of biology is nowhere near complete. For example, in the worm, C. elegans, less than half of the predicted proteins have a known function (Consortium, 1998). The major challenge facing biologists in the next decade will be to ''finish the(More)
The completion and near completion of the sequencing phase of genome projects has ushered in the age of proteomics, the study of all gene products in an organism. This flood of sequence information coupled with recent advances in molecular and structural biology have led to the concept of 'struc-tural proteomics' or 'structural genomics', the determination(More)
Transmission electron microscopy (TEM) observations of graphite tubules (buckytubes) and their derivatives have revealed not only the previously reported buckytube geometries but also additional shapes of the buckytube derivatives. Detailed cross-sectional TEM images reveal the cylindrical cross section of buckytubes and the growth pattern of buckytubes as(More)