Table 2.

Summarizing computational tools for evolutionary conservation based focused library generation.

Approach	Name	Description	Case study examples	URL
Hotspot identification	ConSurf 2010 [35]	The web server performs MSA and calculates evolutionary conservation rate to identify conserved positions in protein or nucleotide sequence/structure.	GAL4 transcription factor [35]	http://consurf.tau.ac.il/
	ConSurf DB [36]	The database provides the predicted results of ConSurf [35] server for known protein structures.	Cytochrome c [36]	http://consurfdb.tau.ac.il/index.php
	JET [38]	The Evolutionary trace based method performs MSA on a set of homologous sequences (from PSI-BLAST) after Gibbs like sampling. The aligned homologous sequences are used to construct distance tree based on Neighbor Joining algorithm. The clustering method is parameterized to identify protein interface or core residues by taking into account the physical-chemical properties and evolutionary conservation.	DNA polymerase I, DNA transferase, allophycocyanin, Leucine dehydrogenase, β-trypsin proteinase, phosphotransferase, human CDC42 gene regulation protein, oncogene protein, signal transduction protein etc [38]	http://www.ihes.fr/∼carbone/data6/legenda.htm
	HotSprint Database [39]	The database provides information about hotspots in protein interface using conservation rate and solvent accessibility of the residues.	Numb phosphotyrosine-binding domain [39]	http://prism.ccbb.ku.edu.tr/hotsprint/
	HotSpot wizard [41]	The web server predicts residue mutability of functionally important residues and visualizes it on protein sequence and structure.	Haloalkane dehalogenase, Phosphotriesterase, 1,3-1,4-b-D-Glucan 4-glucanohydrolase, β-Lactamase [41]	http://loschmidt.chemi.muni.cz/hotspotwizard/
	Selecton [42]	The web server detects selection forces on biologically significant sites in the target protein during evolutionary process.	TRIM5α protein [42]	http://selecton.tau.ac.il/index.html
Protein superfamily based MSA	3DM [37]	The database performs structure based MSA for a protein superfamily with sequence, structural, molecular interaction and mutational information from the literature.	α/β hydrolase fold [53]	http://3dmcsis.systemsbiology.nl/
	The Lipase Engineering Database [43, 54, 55]	The database performs protein superfamily based MSA and annotates functionally relevant amino acid positions with structural and mutational information.	Lipases [43, 54, 55]	http://www.led.uni-stuttgart.de/
	The database of epoxide hydrolases and haloalkane dehalogenase [56]		Epoxide hydrolases and haloalkane dehalogenase [56]	http://www.led.uni-stuttgart.de/
	The Laccase Engineering database [45]		Laccases [45]	http://www.lcced.uni-stuttgart.de/
	The Cytochrome P450 engineering database [57]		Cytochrome P450s [57]	http://www.cyped.uni-stuttgart.de/
	The PHA Depolymerase Engineering Database [44]		Polyhydroxyalkanoates depolymerase [44]	http://www.ded.uni-stuttgart.de/
	The Lactamase Engineering database [46]		Lactamases [46]	http://www.laced.uni-stuttgart.de/
	SHV Lactamase Engineering Database [47]		SHV lactamases [47]	http://www.laced.uni-stuttgart.de/classA/SHVED/
Literature based protein mutant data	PMD [48]	The database provides literature based protein mutant information with structure and functional annotation.		http://pmd.ddbj.nig.ac.jp/∼pmd/pmd.html
	ProTherm [49–51]	The database provides literature based protein mutant information with thermodynamic parameters and experimental conditions integrated with sequence, structure and function annotation.		http://gibk26.bio.kyutech.ac.jp/jouhou/Protherm/protherm.html
	MuteinDB [52]	The database provides literature based protein mutant information, kinetic parameters and experimental conditions integrated with user-friendly and flexible query system to fetch data using reaction name or substrate or inhibitor name or structure and mutations.	Cytochrome P450s [52]	https://muteindb.genome.tugraz.at/muteindb-web-2.0/faces/init/index.seam