Supplementary Material
Characterization of cleavage intermediate and star sites of RM.Tth111II
Zhenyu Zhu*, Shengxi Guan, Derek Robinson, Hanna El Fezzazi, Aine Quimby, 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: http://prodata.swmed.edu/promals3d/promals3d.php).
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,
MTase motifs 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:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
1
1
71
56
141
126
211
196
281
266
351
336
421
406
491
476
561
546
9999999999999999999999999999999999999999999999999999999999999999999999
MLSLLTGGVFRRVKLMNWIDLYTHLKQEVPWFFNSVRLAASQAHNEAEFESRINNAIERLAQKLGVQLLF
---------------MNWIDLYTHLKQEVPWFFNSVRLAASQAHNEAEFESRINNAIERLAQKLGVQLLF
...............MNWIDLYTHLKQEVPWFFNSVRLAASQAHNEAEFESRINNAIERLAQKLGVQLLF
hhh hhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhhh
hhhhhhhhhhhhhhhh
eeee
9999999999999999999999999999999999999999999999999999999999999999999999
REQYTLATGRADAVYNRLVIEYEPPGSLRPNLKHSHTQHAVRQVMNYIEELSRAERHDRDRLLGVVFDGH
REQYTLATGRADAVYNRLVIEYEPPGSLRPNLKHSHTQHAVRQVMNYIEELSRAERHDRDRLLGVVFDGH
REQYTLATGRADAVYNRLVIEYEPPGSLRPNLKHSHTQHAVRQVMNYIEELSRAERHDRDRLLGVVFDGH
eeeeee
hhhhhhhhhhhhhhhhh
hhhh hhhh
9999999999999999999999999999999999999999999999999999999999999999999999
YFIFVRYHEGHWIVEEPLEVNPASCERFLRSLFSLSSGRALIPENLVEDFGSQNDLSRQATRALYHALQG
YFIFVRYHEGHWIVEEPLEVNPASCERFLRSLFSLSSGRALIPENLVEDFGSQNDLSRQATRALYHALQG
YFIFVRYHEGHWIVEEPLEVNPASCERFLRSLFSLSSGRALIPENLVEDFGSQNDLSRQATRALYHALQG
eeeeeee
eee
hhhhhhhhhhhhh
hhhhhhhhhhhh hhhhhhhhhhhhhhh
9999999999999999999999999999999999999999999999999999999999999999999999
HTSDLTARLFVQWQIFFGETAGADAAGGELKHKSELLAFARGMGLRGSRIDMPRFLFALHTYFSFLVKNI
HTSDLTARLFVQWQIFFGETAGADAAGGELKHKSELLAFARGMGLRGSRIDMPRFLFALHTYFSFLVKNI
HTSDLTARLFVQWQIFFGETAGADAAGGELKHKSELLAFARGMGLRGSRIDMPRFLFALHTYFSFLVKNI
hhhhhhhhhhhhh
hhh hhhhhhhhhhhh
hhhhhhhhhhhhhhhhhhh
9999999999999999999999999999999999999999999999999999999999999999999999
ARLVLQAYAGGGLGTTPLTTIANLEGEALRRELQNLESGGLFRTLGLKNLLEGDFFAWYLDAWNPEVEEA
ARLVLQAYAGGGLGTTPLTTIANLEGEALRRELQNLESGGLFRTLGLKNLLEGDFFAWYLDAWNPEVEEA
ARLVLQAYAGGGLGTTPLTTIANLEGEALRRELQNLESGGLFRTLGLKNLLEGDFFAWYLDAWNPEVEEA
hhhhhh
hhh
hhhhhhhhhhhh
hhhh
hhhhhhhhhhh hhhhhh
9999999999999999999999999999999999999999999999999999999999999999999999
LRQVLARLAEYNPATVQDDPHSARDLLKKLYHYLLPRDIRHDLGEFYTPDWLAERLLNQLGEPWFIMPPG
LRQVLARLAEYNPATVQDDPHSARDLLKKLYHYLLPRDIRHDLGEFYTPDWLAERLLNQLGEPWFIMPPG
LRQVLARLAEYNPATVQDDPHSARDLLKKLYHYLLPRDIRHDLGEFYTPDWLAERLLNQLGEPWFIMPPG
hhhhhhhhhhhhh
hhhhhhhhhhhhhhhh
hhhhhhhhhhh hhhhhh
9999999999999999999999 99999999999999999999999999999999999999999999999
NHPPRGLPDKRLLDPACGSGTFLVLAIRALKVNCFLAGFSEADTLEVILNSVVGIDLNPLAVTAARVNYL
NHPPRGLPDKRLLDPACGSGTFPVLAIRALKVNCFLAGFSEADTLEVILNSVVGIDLNPLAVTAARVNYL
NHPPRGLPDKRLLDPACGSGTF.VLAIRALKVNCFLAGFSEADTLEVILNSVVGIDLNPLAVTAARVNYL
eee
hhhhhhhhhhhhhhhh
hhhhhhhhh eeeee hhhhhhhhhhhh
9999999999999999999999999999999999999999999999999999999999999999999999
LAIADLLPYRRREVEIPVYLADSILTPARGEGLFAQNRRILETAVGPLPVPEVINSRAKMERLTDLLEEY
LAIADLLPYRRREVEIPVYLADSILTPARGEGLFAQNRRILETAVGPLPVPEVINSRAKMERLTDLLEEY
LAIADLLPYRRREVEIPVYLADSILTPARGEGLFAQNRRILETAVGPLPVPEVINSRAKMERLTDLLEEY
hhh
hhhhhhhhhhhhhh
999999999999999999999999999999 9 9999999999999999999999999999999999999
VRGDFSTEAFLARAKKEIPDLADALHADEVLTELYERLRDLHRQGLDGIWARVLKNAFMPLFLEPFDYVV
VRGDFSTEAFLARAKKEIPDLADALHADEVITGLYERLRDLHRQGLDGIWARVLKNAFMPLFLEPFDYVV
70
55
140
125
210
195
280
265
350
335
420
405
490
475
560
545
630
615
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
Conservation:
TthHB27I_CAARCA
Tth111II_CAARCA
Consensus_aa:
Consensus_ss:
VRGDFSTEAFLARAKKEIPDLADALHADEVlT.LYERLRDLHRQGLDGIWARVLKNAFMPLFLEPFDYVV
h
hhhhhhhhhhhhhhhh
hhhhhhhhhhhhhhhhhhhhhhhhhhh
eeeee
631
616
701
686
771
756
841
826
911
896
981
966
9999999999999999999999999999999999999999999999999999999999999999999999
GNPPWINWESLPQAYREQTAELWTCYGLFVHSGMDTILGKGKKDASTLMTYAVADRFLKEGGKLGFLITQ
GNPPWINWESLPQAYREQTAELWTCYGLFVHSGMDTILGKGKKDASTLMTYAVADRFLKEGGKLGFLITQ
GNPPWINWESLPQAYREQTAELWTCYGLFVHSGMDTILGKGKKDASTLMTYAVADRFLKEGGKLGFLITQ
hhhhhhhhhhhhhhhhh
hhhhhhhhhhhhhhh
eeeeee
999999999999999999999999999999999999999999999999999999 999999999999999
SVWKTGAGQGFRRFRIGENGPHLRVLHVDDLSSLQVFEGASTRTSAFVLQKGRPTRYPVPYTYWKKTTKG
SVWKTGAGQGFRRFRIGENGPHLRVLHVDDLSSLQVFEGASTRTSAFVLQKGRPPRYPVPYTYWKKTTKG
SVWKTGAGQGFRRFRIGENGPHLRVLHVDDLSSLQVFEGASTRTSAFVLQKGRPsRYPVPYTYWKKTTKG
hhh
hhhhhhhhhh
eeeeeee
eeeeeeeee
eeeee
99999999999999999999999999999999999999999999 9999999999999999999999999
EGLDYDSTLGEVMEQTKRLRFHAVPVDPDDLTSPWLTARRRALYAVRKVLGTSEYRAYEGANSGGANGIY
EGLDYDSTLGEVMEQTKRLRFHAVPVDPDDLTSPWLTARRRALYSVRKVLGTSEYRAYEGANSGGANGIY
EGLDYDSTLGEVMEQTKRLRFHAVPVDPDDLTSPWLTARRRALYtVRKVLGTSEYRAYEGANSGGANGIY
hhhhhhh hhhhhhh
hhhhhh
ee
ee
9999999999999999999999999999999999999999999999999999999999999999999999
WLEILAERPDGLVVVRNVTEGAKREVEGITTELEPDLLYPLLRGRDVRRWYAQPSLHILMVQDPKTRRGI
WLEILAERPDGLVVVRNVTEGAKREVEGITTELEPDLLYPLLRGRDVRRWYAQPSLHILMVQDPKTRRGI
WLEILAERPDGLVVVRNVTEGAKREVEGITTELEPDLLYPLLRGRDVRRWYAQPSLHILMVQDPKTRRGI
ee
hhhhhhhhhh
hhhhhhhh
eee
9999999999999999999999999999999999999999999999999999999999999999999999
DEQVLQKRYPKTWAYLKRFEAVLRERSGFRRYFTRKDRNGRMVETGPFYSMFNVGDYTFAPWKVVWRYVA
DEQVLQKRYPKTWAYLKRFEAVLRERSGFRRYFTRKDRNGRMVETGPFYSMFNVGDYTFAPWKVVWRYVA
DEQVLQKRYPKTWAYLKRFEAVLRERSGFRRYFTRKDRNGRMVETGPFYSMFNVGDYTFAPWKVVWRYVA
hhhhhhhhhhhhhhhhhhhhhhhh
eeeee
700
685
770
755
840
825
910
895
980
965
9999999999999999999999999999999999999999999999999999999999999999999999
SDFIVAVVGPASDEKPVVPNEKLMLVPVEDDNEAFYLCGVLNSSPIRFAVQSFFVQTQIAPHVLQKLCIP 1050
SDFIVAVVGPASDEKPVVPNEKLMLVPVEDDNEAFYLCGVLNSSPIRFAVQSFFVQTQIAPHVLQKLCIP 1035
SDFIVAVVGPASDEKPVVPNEKLMLVPVEDDNEAFYLCGVLNSSPIRFAVQSFFVQTQIAPHVLQKLCIP
eeeee
eeee eeeee
hhhhhhhhhhh hhhhhhhhhh
ee
hh
Conservation:
TthHB27I_CAARCA 1051
Tth111II_CAARCA 1036
Consensus_aa:
Consensus_ss:
9999999999999999999999999999999999999999999999999999999999999999999999
RYEPNTDHQNRIAHLSRRAHELAPAAYNGDKAARAELRRVEEEIDRAAAQLWGLTEEELAEIRRSLEELR 1120
RYEPNTDHQNRIAHLSRRAHELAPAAYNGDKAARAELRRVEEEIDRAAAQLWGLTEEELAEIRRSLEELR 1105
RYEPNTDHQNRIAHLSRRAHELAPAAYNGDKAARAELRRVEEEIDRAAAQLWGLTEEELAEIRRSLEELR
hhhhhhhhhhhhhhhhhhhh
hhhhhhhhhhhhhhhhhhhhhh
hhhhhhhhhhhhhh
Conservation:
TthHB27I_CAARCA 1121
Tth111II_CAARCA 1106
Consensus_aa:
Consensus_ss:
9
G 1121
G 1106
G