Supplementary Material
Characterization of cleavage intermediate and star sites of RM.Tth111II
Zhenyu Zhu*, Shengxi Guan, Derek Robinson, Hanna El Fezzazi, AineQuimby, and
Shuang-yong Xu*
New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA
Supplementary Figure 1.
Color-coded functional domains and secondary structure prediction and PROMALS3D alignment (computer server: Tth111II and TthHB27I amino acid sequences were used in the alignment. The exact boundaries of the functional domains remain to be determined by structure analysis and experimentation.
Blue=endonuclease catalytic domain (rough boundary: aa 1-146)
Green=alpha helical domain (rough boundary: aa 147-374)
Orange=N6-adenine methyltransferase group (rough boundary 375-726, MTasemotifs X (FYTP), I (GSGT), VIII (VFEGAS), underlined aa blocks)
Dark blue=specificity domain (rough boundary 727-1106)
Secondary structure (ss) prediction: h, helix; e, sheet. 9=high probability of the secondary structure prediction (PROMALS3D)
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 1 MLSLLTGGVFRRVKLMNWIDLYTHLKQEVPWFFNSVRLAASQAHNEAEFESRINNAIERLAQKLGVQLLF 70
Tth111II_CAARCA 1 ------MNWIDLYTHLKQEVPWFFNSVRLAASQAHNEAEFESRINNAIERLAQKLGVQLLF 55
Consensus_aa: ...... MNWIDLYTHLKQEVPWFFNSVRLAASQAHNEAEFESRINNAIERLAQKLGVQLLF
Consensus_ss: hhh hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhh eeee
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 71 REQYTLATGRADAVYNRLVIEYEPPGSLRPNLKHSHTQHAVRQVMNYIEELSRAERHDRDRLLGVVFDGH 140
Tth111II_CAARCA 56 REQYTLATGRADAVYNRLVIEYEPPGSLRPNLKHSHTQHAVRQVMNYIEELSRAERHDRDRLLGVVFDGH 125
Consensus_aa: REQYTLATGRADAVYNRLVIEYEPPGSLRPNLKHSHTQHAVRQVMNYIEELSRAERHDRDRLLGVVFDGH
Consensus_ss: eeeeee hhhhhhhhhhhhhhhhh hhhhhhhh
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 141 YFIFVRYHEGHWIVEEPLEVNPASCERFLRSLFSLSSGRALIPENLVEDFGSQNDLSRQATRALYHALQG 210
Tth111II_CAARCA 126 YFIFVRYHEGHWIVEEPLEVNPASCERFLRSLFSLSSGRALIPENLVEDFGSQNDLSRQATRALYHALQG 195
Consensus_aa: YFIFVRYHEGHWIVEEPLEVNPASCERFLRSLFSLSSGRALIPENLVEDFGSQNDLSRQATRALYHALQG
Consensus_ss: eeeeeee eee hhhhhhhhhhhhh hhhhhhhhhhhh hhhhhhhhhhhhhhh
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 211 HTSDLTARLFVQWQIFFGETAGADAAGGELKHKSELLAFARGMGLRGSRIDMPRFLFALHTYFSFLVKNI 280
Tth111II_CAARCA 196 HTSDLTARLFVQWQIFFGETAGADAAGGELKHKSELLAFARGMGLRGSRIDMPRFLFALHTYFSFLVKNI 265
Consensus_aa: HTSDLTARLFVQWQIFFGETAGADAAGGELKHKSELLAFARGMGLRGSRIDMPRFLFALHTYFSFLVKNI
Consensus_ss: hhhhhhhhhhhhh hhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhhh
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 281 ARLVLQAYAGGGLGTTPLTTIANLEGEALRRELQNLESGGLFRTLGLKNLLEGDFFAWYLDAWNPEVEEA 350
Tth111II_CAARCA 266 ARLVLQAYAGGGLGTTPLTTIANLEGEALRRELQNLESGGLFRTLGLKNLLEGDFFAWYLDAWNPEVEEA 335
Consensus_aa: ARLVLQAYAGGGLGTTPLTTIANLEGEALRRELQNLESGGLFRTLGLKNLLEGDFFAWYLDAWNPEVEEA
Consensus_ss: hhhhhh hhh hhhhhhhhhhhh hhhh hhhhhhhhhhh hhhhhh
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 351 LRQVLARLAEYNPATVQDDPHSARDLLKKLYHYLLPRDIRHDLGEFYTPDWLAERLLNQLGEPWFIMPPG 420
Tth111II_CAARCA 336 LRQVLARLAEYNPATVQDDPHSARDLLKKLYHYLLPRDIRHDLGEFYTPDWLAERLLNQLGEPWFIMPPG 405
Consensus_aa: LRQVLARLAEYNPATVQDDPHSARDLLKKLYHYLLPRDIRHDLGEFYTPDWLAERLLNQLGEPWFIMPPG
Consensus_ss: hhhhhhhhhhhhh hhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhh
Conservation: 9999999999999999999999 99999999999999999999999999999999999999999999999
TthHB27I_CAARCA 421 NHPPRGLPDKRLLDPACGSGTFLVLAIRALKVNCFLAGFSEADTLEVILNSVVGIDLNPLAVTAARVNYL 490
Tth111II_CAARCA 406 NHPPRGLPDKRLLDPACGSGTFPVLAIRALKVNCFLAGFSEADTLEVILNSVVGIDLNPLAVTAARVNYL 475
Consensus_aa: NHPPRGLPDKRLLDPACGSGTF.VLAIRALKVNCFLAGFSEADTLEVILNSVVGIDLNPLAVTAARVNYL
Consensus_ss: eee hhhhhhhhhhhhhhhh hhhhhhhhh eeeee hhhhhhhhhhhh
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 491 LAIADLLPYRRREVEIPVYLADSILTPARGEGLFAQNRRILETAVGPLPVPEVINSRAKMERLTDLLEEY 560
Tth111II_CAARCA 476 LAIADLLPYRRREVEIPVYLADSILTPARGEGLFAQNRRILETAVGPLPVPEVINSRAKMERLTDLLEEY 545
Consensus_aa: LAIADLLPYRRREVEIPVYLADSILTPARGEGLFAQNRRILETAVGPLPVPEVINSRAKMERLTDLLEEY
Consensus_ss: hhh hhhhhhhhhhhhhh
Conservation: 999999999999999999999999999999 9 9999999999999999999999999999999999999
TthHB27I_CAARCA 561 VRGDFSTEAFLARAKKEIPDLADALHADEVLTELYERLRDLHRQGLDGIWARVLKNAFMPLFLEPFDYVV 630
Tth111II_CAARCA 546 VRGDFSTEAFLARAKKEIPDLADALHADEVITGLYERLRDLHRQGLDGIWARVLKNAFMPLFLEPFDYVV 615
Consensus_aa: VRGDFSTEAFLARAKKEIPDLADALHADEVlT.LYERLRDLHRQGLDGIWARVLKNAFMPLFLEPFDYVV
Consensus_ss: h hhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhhhhhhhhhhh eeeee
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 631 GNPPWINWESLPQAYREQTAELWTCYGLFVHSGMDTILGKGKKDASTLMTYAVADRFLKEGGKLGFLITQ 700
Tth111II_CAARCA 616 GNPPWINWESLPQAYREQTAELWTCYGLFVHSGMDTILGKGKKDASTLMTYAVADRFLKEGGKLGFLITQ 685
Consensus_aa: GNPPWINWESLPQAYREQTAELWTCYGLFVHSGMDTILGKGKKDASTLMTYAVADRFLKEGGKLGFLITQ
Consensus_ss: hhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhh eeeeee
Conservation: 999999999999999999999999999999999999999999999999999999 999999999999999
TthHB27I_CAARCA 701 SVWKTGAGQGFRRFRIGENGPHLRVLHVDDLSSLQVFEGASTRTSAFVLQKGRPTRYPVPYTYWKKTTKG 770
Tth111II_CAARCA 686 SVWKTGAGQGFRRFRIGENGPHLRVLHVDDLSSLQVFEGASTRTSAFVLQKGRPPRYPVPYTYWKKTTKG 755
Consensus_aa: SVWKTGAGQGFRRFRIGENGPHLRVLHVDDLSSLQVFEGASTRTSAFVLQKGRPsRYPVPYTYWKKTTKG
Consensus_ss: hhh hhhhhhhhhh eeeeeee eeeeeeeee eeeee
Conservation: 99999999999999999999999999999999999999999999 9999999999999999999999999
TthHB27I_CAARCA 771 EGLDYDSTLGEVMEQTKRLRFHAVPVDPDDLTSPWLTARRRALYAVRKVLGTSEYRAYEGANSGGANGIY 840
Tth111II_CAARCA 756 EGLDYDSTLGEVMEQTKRLRFHAVPVDPDDLTSPWLTARRRALYSVRKVLGTSEYRAYEGANSGGANGIY 825
Consensus_aa: EGLDYDSTLGEVMEQTKRLRFHAVPVDPDDLTSPWLTARRRALYtVRKVLGTSEYRAYEGANSGGANGIY
Consensus_ss: hhhhhhh hhhhhhh hhhhhh ee ee
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 841 WLEILAERPDGLVVVRNVTEGAKREVEGITTELEPDLLYPLLRGRDVRRWYAQPSLHILMVQDPKTRRGI 910
Tth111II_CAARCA 826 WLEILAERPDGLVVVRNVTEGAKREVEGITTELEPDLLYPLLRGRDVRRWYAQPSLHILMVQDPKTRRGI 895
Consensus_aa: WLEILAERPDGLVVVRNVTEGAKREVEGITTELEPDLLYPLLRGRDVRRWYAQPSLHILMVQDPKTRRGI
Consensus_ss: ee hhhhhhhhhh hhhhhhhh eee
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 911 DEQVLQKRYPKTWAYLKRFEAVLRERSGFRRYFTRKDRNGRMVETGPFYSMFNVGDYTFAPWKVVWRYVA 980
Tth111II_CAARCA 896 DEQVLQKRYPKTWAYLKRFEAVLRERSGFRRYFTRKDRNGRMVETGPFYSMFNVGDYTFAPWKVVWRYVA 965
Consensus_aa: DEQVLQKRYPKTWAYLKRFEAVLRERSGFRRYFTRKDRNGRMVETGPFYSMFNVGDYTFAPWKVVWRYVA
Consensus_ss: hhhhhhhhhhhhhhhhhhhhhhhh eeeee
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 981 SDFIVAVVGPASDEKPVVPNEKLMLVPVEDDNEAFYLCGVLNSSPIRFAVQSFFVQTQIAPHVLQKLCIP 1050
Tth111II_CAARCA 966 SDFIVAVVGPASDEKPVVPNEKLMLVPVEDDNEAFYLCGVLNSSPIRFAVQSFFVQTQIAPHVLQKLCIP 1035
Consensus_aa: SDFIVAVVGPASDEKPVVPNEKLMLVPVEDDNEAFYLCGVLNSSPIRFAVQSFFVQTQIAPHVLQKLCIP
Consensus_ss: eeeee eeee eeeee hhhhhhhhhhh hhhhhhhhhh ee hh
Conservation: 9999999999999999999999999999999999999999999999999999999999999999999999
TthHB27I_CAARCA 1051 RYEPNTDHQNRIAHLSRRAHELAPAAYNGDKAARAELRRVEEEIDRAAAQLWGLTEEELAEIRRSLEELR 1120
Tth111II_CAARCA 1036 RYEPNTDHQNRIAHLSRRAHELAPAAYNGDKAARAELRRVEEEIDRAAAQLWGLTEEELAEIRRSLEELR 1105
Consensus_aa: RYEPNTDHQNRIAHLSRRAHELAPAAYNGDKAARAELRRVEEEIDRAAAQLWGLTEEELAEIRRSLEELR
Consensus_ss: hhhhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhhhhhhhhhh hhhhhhhhhhhhhh
Conservation: 9
TthHB27I_CAARCA 1121 G 1121
Tth111II_CAARCA 1106 G 1106
Consensus_aa: G
Consensus_ss: