Table 1. Software, databases and selected protocols employed in sgTarget’s annotation pipeline W226 Nucleic Acids Research, 2006, Vol. 34, Web Server issue targets. The pipeline consists of a set of bioinformatics methods that were selected and incorporated into the resource’s framework, as follows: Methods to predict protein fold, function and prevalence. These help to identify targets, such as proteins for which fold predictions cannot yet be established, those with unknown functions, or ORFan proteins. Assessment of known protein expression and crystallization issues. Nucleotide sequence based calculations deter- mine the encoding gene’s GC content, codon usage and its compatibility with that of the host expression system (the Codon Adaptation Index). These metrics can highlight potential problems for protein expression. Similarly, sequence based prediction of protein instability, solubility and half-life can identify issues for high throughput structure determination. Assessment of known protein structure issues. Protein sequence based calculations predict the locations of intrinsi- cally disordered, fibrous or transmembrane regions. The presence of these features can pose challenges for structure determination. The majority of protocols employed by the annotation pipeline use established bioinformatics methods and databases (listed in Table 1). A novel procedure for the identiﬁcation of intrinsically disordered regions was developed (5) and is described brieﬂy below. In addition, tailored thresholds were established for GC content (between 26.9 to 66.8% for the expression host Escherichia coli), Codon Adaptation Index (above 0.084 for expression in E.coli, and above 0.357 for high levels of expression) and E-value cutoffs to assess the structural signiﬁcance of BLAST alignments (two cutoffs are employed by the resource: 2.07 · 10 11, a conservative threshold and 2.15 · 10 4, a ‘natural’ threshold with a false positive rate of 0.2%). Identification of intrinsically disordered regions Intrinsically disordered domains can cause a multitude of adverse effects in structural determination studies, including puriﬁcation difﬁculties due to hypersensitivity to protease digestion, missing electron density due to incoherent X-ray scattering, hindered crystallization, extreme broadening of side chain NMR peaks and lack of chemical shift dispersion of NMR backbone data. Some of these segments may become ordered upon interaction with binding partners to perform speciﬁc functions (6). Their structural characterization would, however, be difﬁcult even if prior knowledge of the required cofactors was available. The annotation pipeline employs the charge-hydrophobicity phase-space boundary of Uversky et al. (7), complemented by the putative lower bound complexity threshold of Romero and colleagues (8), to predict regions of intrinsic disorder. The low-complexity detection software SEG isolates subsequences with high or low-complexity on the basis of information content (9). In sgTarget, SEG is employed to detect any subsequences of at least 45 residues and a complexity value lower than 2.90. Such regions are annotated as probable non-globular protein stretches. For the remaining subsequences the mean hydrophobicity [the sum of the normalized hydrophobicities from (10) divided by the Software CodonWa BLAST (18) InterProScan (19) SEG (9) TMHMM (21) Database PDB SEQRES (22) InterPro (23) GO (24) NRDB Taxonomyb Protocol Instability index (25) Estimate half-life using the N-end rule (26) Wilkinson–Harrison solubility index (27,28) Application Calculate the relative conformance of a gene to an organism’s genome (the Codon Adaptation Index) Perform local protein sequence similarity searches against PDB and NRDB sequences Run sequence comparison methods required to search the InterPro database (as well as NCOILS (20) to identify coiled-coil domains) Detect and isolate subsequences with high or low-complexity Predict the location and topology of protein transmembrane regions Description Protein sequences derived from the SEQRES card of PDB files Integrated collection of the protein domain family databases (Pfam, PRINTS, ProDom, PROSITE, SMART, TIGRFAMs and PANTHER) Function ontology database with mappings to InterPro Collection of protein sequence databases (PIR, SWISS-PROT, TrEMBL and PDB SEQRES) Taxonomical classification of organisms cross-referenced by NRDB Description & Application The instability index is a length-scaled measure of the occurrence of all dipeptides in a protein sequence. Guruprasad and colleagues found a correlation between this measure and protein stability: in general, stable proteins have instability indices smaller than 40. Estimates of in vivo half-life for proteolysis of proteins in prokaryotes can be made by the N-end rule. This considers the presence of a destabilizing N-terminal residue that provides an N-degron degradation signal. The revised Wilkinson–Harrison statistical solubility model depends on two parameters: the fraction of residues with a high index for forming turns and the approximate average charge of the protein in vivo. This model has been shown to be useful in the selection of proteins with high solubility. hHi ¼ h R i þ 1:151 2:785 1 aCodonW (http://codonw.sourceforge.net/). bTaxonomy (http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html). number of residues] and the mean net charge at pH 7.0 are calculated, and used in Equation 1, to predict if a subsequence is likely to be intrinsically disordered. Uversky and colleagues found that disordered proteins have low overall hydrophobi- city and high net charge, always falling below the boundary: where hHi is the mean hydrophobicity and hRi is the mean net charge (7,11). The performance of sgTarget’s disorder prediction method on the CASP5 disorder benchmark was evaluated (12). sgTarget’s disorder predictions for those targets that are least related to a protein with known structure, achieved an accuracy of 0.77 (where accuracy is the arithmetic mean of sensitivity and speciﬁcity measured on a per residue basis), which compares favorably to previously reported methods.