Supplementary material belonging to
EZ-ASSIGN, a program for exhaustive NMR backbone assignments of large proteins
from complete or incomplete triple-resonance data
Erik R.P. Zuiderweg, Ireena Bagai, Paolo Rossi, and Eric B. Bertelsen.
Table S1. Amino acid translationsReduced / 7-word / 6-word / 5-word / Oxydized / 7-word / 6-word / 5-word
A / A / A / A / A / A / A / A
G / G / G / G / G / G / G / G
D F I L N Y / O / O / B / C D F I L N Y / O / O / B
C E H K M Q R V W / B / B / CE H K M Q R V W / B / B
S / S / Z / Z / S / S / Z / Z
T / T / T / T
P / P / P / P / P / P / P / P
Based on the statistics in the BMRB (see also REF)
Table S2: Adapted BMRB statistics for 13Ca and 13Cb chemical shifts used in this work
group / res / Ca center / Ca S.D. / Cb center / Cb S.D.
A / A / 53.16 / 2.04 / 18.93 / 1.88 a /4.0b
G / G / 45.36 / 1.35
P / P / 63.27 / 1.64 / 31.75 / 1.25
O / D / 54.62 / 2.09 / 40.8 / 1.65
F / 58.22 / 2.7 / 39.91 / 2.09
I / 61.59 / 2.78 / 38.55 / 2.08
L / 55.62 / 2.2 / 42.26 / 1.92
N / 53.51 / 1.96 / 38.63 / 1.78
Y / 58.06 / 2.6 / 39.28 / 2.2
Cox / 55.5 / 2.5 / 40.7 / 3.8
B / Cred / 60. / 3.43 / 28. / 2.66
E / 57.43 / 2.14 / 30.0 / 1.79
H / 56.46 / 2.47 / 30.2 / 2.17
K / 56.9 / 2.26 / 32.74 / 1.86
M / 56.19 / 2.27 / 33.01 / 2.29
Q / 56.53 / 2.19 / 29.18 / 1.92
R / 56.83 / 2.39 / 30.6 / 1.84
V / 62.46 / 2.96 / 32.63 / 1.83
W / 57.66 / 2.62 / 30.02 / 2.08
Z / S / 58.64 / 2.18 / 63.82 / 1.61
T / 62.13 / 2.72 / 69.61 / 2.05
a) BMRB value
b) optimized on Maleate synthase data using EZ-ASSIGN
Table S3A
Number of correct minus incorrect assignments for Malate Synthase while assigning unique nona peptides
a x 1 / a x 1.5 / a x 2 / a x 2.5 / a x 3.0 / a x 3.5
b x 1 / 0 / 44 / 44 / 54 / 54 / 54
b x 1.5 / 19 / 191 / 293 / 334 / 334 / 334
b x 2.0 / 20 / 274 / 443 / 484 / 488 / 488
b x 2.5 / 34 / 341 / 546 / 597 / 601 / 601
b x 3.0 / 34 / 353 / 552 / 612 / 616 / 607
b x3.5 / 34 / 361 / 561 / 621 / 625 / 616
b x 4.0 / 34 / 361 / 561 / 612 / 625 / 634
Required rung matches: 2; Ca, Cb and CO rung tolerances: +/- 0.05 ppm.
Word length: 7.
Table S3B
Number of correct minus incorrect assignments for Malate Synthase while assigning unique hexa peptides
a x 1 / a x 1.5 / a x 2 / a x 2.5 / a x 3.0 / a x 3.5
b x 1 / 14 / 109 / 131 / 149 / 155 / 155
b x 1.5 / 46 / 269 / 358 / 398 / 400 / 393
b x 2.0 / 61 / 373 / 481 / 521 / 507 / 488
b x 2.5 / 81 / 438 / 507 / 541 / 496 / 433
b x 3.0 / 81 / 418 / 475 / 475 / 393 / 235
b x / 81 / 401 / 416 / 338 / 180 / -88
b x 4.0 / 81 / 359 / 255 / 78 / -327 / -774
Required rung matches: 2; Ca, Cb and CO rung tolerances: +/- 0.05 ppm.
Word length: 7.
Table S3C
Number of correct minus incorrect assignments for Malate Synthase while assigning unique tetra peptides
a x 1 / a x 1.5 / a x 2 / a x 2.5 / a x 3.0 / a x 3.5
b x 1 / 38 / 153 / 182 / 186 / 188 / 176
b x 1.5 / 65 / 291 / 360 / 352 / 340 / 310
b x 2.0 / 85 / 334 / 396 / 376 / 308 / 257
b x 2.5 / 87 / 356 / 384 / 303 / 82 / -66
b x 3.0 / 78 / 325 / 269 / 69 / -351 / -669
b x / 62 / 246 / 91 / -294 / -1016 / -1628
b x 4.0 / 41 / 82 / -258 / -924 / -2036 / -3001
Required rung matches: 2; Ca, Cb and CO rung tolerances: +/- 0.05 ppm.
Word length: 7.
Table S3D
Number of correct minus incorrect assignments for Malate Synthase while assigning unique di peptides
a x 1 / a x 1.5 / a x 2 / a x 2.5 / a x 3.0 / a x 3.5
b x 1 / 11 / 17 / 17 / 13 / 9 / -5
b x 1.5 / 13 / 21 / 21 / 17 / 13 / -9
b x 2.0 / 17 / 19 / 15 / 8 / 0 / -24
b x 2.5 / 17 / 11 / 5 / -14 / -69 / -117
b x 3.0 / 11 / -9 / -31 / -70 / -157 / -234
b x / -1 / -31 / -81 / -138 / -255 / -357
b x 4.0 / -13 / -60 / -130 / -209 / -366 / -494
Required rung matches: 2; Ca, Cb and CO rung tolerances: +/- 0.05 ppm.
Word length: 7.
Table S4 Effect of sequence translation code
Non-proline containing peptides in malate synthase (723 residues) / Found
/assigned
/ratio / Found
/assigned
/ratio / Found
/assigned
/ratio / Found
/assigned
length / Unique in 5 letter / Unique in 6 letter / Unique in 7 letter / Unique in 20 letter code
2 / 0/0/* / 0/0/* / 4/93/2.3 / 105/10937
3 / 10/31/3.0 / 25/175/7.0 / 53/287/5.4 / 554/14406
4 / 41/122/3.0 / 139/520/3.7 / 187/654/3.5 / 624/5651
5 / 95/155/1.6 / 334/769/2.3 / 359/807/2.2 / 601/2089
6 / 157/183/1.2 / 453/669/1.5 / 462/682/1.5 / 573/1005
7 / 222/217/1.0 / 506/549/1.1 / 510/553/1.1 / 547/630
8 / 290/262/<1.0 / 506/478/<1.0 / 506/478/<1.0 / 522/502
9 / 338/300/<1.0 / 487/439/<1.0 / 487/439/<1.0 / 497/455
10 / 386/317/<1.0 / 490/411/<1.0 / 490/411/<1.0 / 496/421
Other conditions:
Ca multiplier 2.5 Cb multiplier 3.0000000 required_rungs 2
Onso see Table S1.
Table S5. Number of correct/wrong assignments for complete malate synthase data as function of peptide search length and rung tolerancea
Rung toleranceb
(ppm) / 9-pep / 8-pep / 7-pep / 6-pep / 5-pep / 4-pep / 3-pep / 2-pep
0.02 / 623/2 / 638/34 / 625/32 / 606/65 / 559/52 / 434/38 / 214/28 / 31/10
0.05 / 623/2 / 638/34 / 625/32 / 606/65 / 559/52 / 434/38 / 214/28 / 31/10
0.1 / 623/50 / 638/71 / 625/84 / 606/108 / 559/105 / 434/63 / 215/37 / 31/14
0.15 / 623/160 / 638/182 / 625/212 / 606/199 / 561/199 / 434/137 / 215/47 / 31/17
a) the range multipliers for the different peptide search lengths were varied according to Table 3a
b) the tolerances were equal for Ca, Cb and CO
c) two rungs match when they are within the sum of their tolerances
Expected / ALL / SN>20 / SN>30 / SN>40 / SN>50 / SN>60
Corr ASSIGS / 370 / 296 / 236 / 124 / 84 / 41 / 11
HNCA / 740 / 584 / 543 / 461 / 406 / 360 / 315
HNCOCA / 370 / 293 / 254 / 176 / 129 / 102 / 82
HNCACB / 1447 / 1168 / 963 / 752 / 623 / 522 / 435
HNCOCACB / 707 / 524 / 420 / 291 / 217 / 162 / 120
HNCO / 370 / 294 / 285 / 285 / 285 / 284 / 276
HNCACO / 740 / 561 / 489 / 429 / 373 / 343 / 310
CA(i)-rungs / 370 / 291 / 289 / 285 / 277 / 258 / 233
CA(i-1)-rungs / 370 / 293 / 254 / 176 / 129 / 102 / 82
CB(i)-rungs / 337 / 260 / 256 / 239 / 211 / 181 / 144
CB(i-1)-rungs / 337 / 231 / 166 / 115 / 88 / 60 / 38
CO(i)-rungs / 370 / 267 / 204 / 144 / 88 / 59 / 34
CO(i-1)-rungs / 370 / 294 / 285 / 285 / 285 / 284 / 276
CA-conn / 370 / 252 / 219 / 163 / 116 / 91 / 72
CB-conn / 337 / 196 / 147 / 102 / 76 / 50 / 31
CO-conn / 370 / 234 / 177 / 132 / 80 / 52 / 30
3-rung / 337 / 184 / 113 / 69 / 39 / 21 / 9
2-rung / 33 / 59 / 77 / 62 / 57 / 40 / 28
1-rung / 0 / 12 / 42 / 51 / 41 / 50 / 50
Corr ASSIGS / 296 / 236 / 124 / 84 / 41 / 11
Table S7 Number of cross peaks in the spectra of a 351-residue domain of the type III ATPase.
expected / ALL / Deg1 / Deg2 / Deg3 / Deg4
Corr ASSIGS / 333 / 281 / 179 / 103 / 59 / 28
HNCA / 666 / 613 / 585 / 479 / 418 / 370
HNCOCA / 333 / 311 / 286 / 185 / 141 / 104
HNCACB / 1295 / 1099 / 998 / 754 / 623 / 516
HNCOCACB / 629 / 488 / 413 / 265 / 205 / 146
HNCO / 333 / 314 / 303 / 303 / 303 / 303
HNCACO / 666 / 611 / 533 / 477 / 418 / 367
CA(i)-rungs / 333 / 302 / 299 / 294 / 287 / 266
CA(i-1)-rungs / 333 / 311 / 286 / 185 / 141 / 104
CB(i)-rungs / 296 / 249 / 245 / 229 / 202 / 173
CB(i-1)-rungs / 296 / 177 / 127 / 80 / 64 / 42
CO(i)-rungs / 333 / 297 / 230 / 174 / 115 / 64
CO(i-1)-rungs / 333 / 314 / 303 / 303 / 303 / 303
CA-conn / 333 / 274 / 251 / 158 / 119 / 83
CB-conn / 296 / 141 / 106 / 61 / 46 / 30
CO-conn / 333 / 271 / 205 / 153 / 104 / 55
3-rung / 296 / 131 / 86 / 43 / 30 / 19
2-rung / 37 / 137 / 112 / 77 / 49 / 23
1-rung / 0 / 19 / 80 / 89 / 81 / 65
Corr ASSIGS / 333 / 281 / 179 / 103 / 59 / 28
Table S8.
EZ-ASSIG / HAND ASSIGNCA_i_1 / CA_i / CB_i_1 / CB_i / CO_i_1 / CO_i / CA_i_1 / CA_i / CB_i_1 / CB_i / CO_i_1 / CO_i
G / 1 / G / 1
A / 2 / A / 2 / A2 / 18.78
H / 3 / H / 3
L / 31 / L31 / 45.97 / 53.20 / 40.65 / 173.47 / 174.91 / L / 31 / L31 / 45.97 / 53.20 / 40.65 / 173.47 / 174.91
P / 32 / P / 32
A / 33 / A / 33 / A33 / 62.91 / 51.49 / 32.01 / 16.79 / 176.29 / 175.20
P / 34 / P / 34
P / 46 / P / 46
F / 47 / F47 / 63.13 / 58.45 / 31.75 / 40.05 / 176.41 / 174.98 / F / 47
N / 48 / N48 / 58.44 / 50.84 / 39.94 / 38.95 / 175.03 / 174.02 / N / 48
P / 49 / P / 49
V / 106 / V / 106 / V106 / 33.92
G / 107 / G107 / 60.48 / 44.67 / 174.27 / 171.16 / G / 107 / G107 / 60.48 / 44.67 / 174.27 / 171.16
L / 108 / L108 / 44.74 / 53.31 / 171.12 / 174.47 / L / 108 / L108 / 44.74 / 53.31 / 171.12 / 174.47
A / 150 / A150 / 47.61 / 55.37 / 28.70 / 19.89 / 174.36 / 180.33 / A / 150 / A150 / 47.61 / 55.37 / 28.70 / 19.89 / 174.36 / 180.33
A / 151 / A / 151 / A151 / 55.29 / 55.42 / 180.35 / 179.80
Y / 152 / Y / 152 / Y152 / 55.34 / 57.63 / 37.88 / 179.67 / 176.98
A / 153 / L294 / 179.41 / 178.54 / A / 153 / A153 / 57.77 / 55.86 / 177.06 / 178.71
T / 154 / T154 / 55.79 / 68.10 / 178.62 / T / 154 / T154 / 55.79 / 68.10 / 178.62
A / 157 / A157 / 64.74 / 56.11 / 17.92 / 178.21 / 178.60 / A / 157 / A157 / 64.74 / 56.11 / 17.92 / 178.21 / 178.60
E / 158 / E / 158 / E158 / 56.08 / 60.16 / 178.67 / 177.51
D / 159 / L290 59.89 57.90 29.87 40.93 179.63 179.46
F291 57.94 61.92 179.40 178.77
K292 61.94 61.61 39.45 31.82 178.83 179.07
Q293 61.52 59.67 179.14 179.41 / D / 159 / D159 / 60.12 / 58.29 / 177.62 / 178.45
F / 160 / F / 160 / F160 / 58.33 / 63.08 / 178.54
R / 161 / R / 161 / R161 63.08 178.61 180.92
D162 59.28 57.63 39.33 180.91 177.76
R163 57.45 57.26 29.92 177.74 176.30
G164 57.14 44.92 29.96 176.44 173.70
Q165 55.16 30.68 173.75 173.97
H166 55.17 56.17 30.58 30.26 173.99 175.54
D / 162 / D / 162
R / 163 / R / 163
G / 164 / G189 / 61.72 / 45.59 / 176.76 / 174.34 / G / 164
Q / 165 / E190 / 45.71 / 54.76 / 30.29 / 174.46 / 174.12 / Q / 165
H / 166 / H / 166
V / 167 / F47 / 63.13 / 58.45 / 31.75 / 40.05 / 176.41 / 174.98 / V / 167
L / 168 / N48 / 58.44 / 50.84 / 39.94 / 38.95 / 175.03 / 174.02 / L / 168
L / 169 / L / 169
I / 170 / I / 170
A / 187 / A187 / 57.63 / 54.84 / 17.95 / 181.28 / 179.55 / A / 187 / A187 / 57.63 / 54.84 / 17.95 / 181.28 / 179.55
I / 188 / L208 / 54.62 / 18.14 / 42.19 / 179.51 / 179.84 / I / 188 / I188 / 54.81 / 61.78 / 38.22 / 179.55
G / 189 / G / 189 / G189 / 61.72 / 45.59 / 176.76 / 174.34
E / 190 / E / 190 / E190 / 45.71 / 54.76 / 30.29 / 174.46 / 174.12
K / 204 / K204 / 53.99 / 57.66 / 32.02 / 180.09 / 178.92 / K / 204 / K204 / 53.99 / 57.66 / 32.02 / 180.09 / 178.92
L / 205 / E183 / 57.62 / 60.40 / 179.14 / 180.13 / L / 205 / L205 / 57.70 / 60.61 / 38.69 / 178.65 / 174.72
P / 206 / P / 206
A / 207 / A / 207 / A207 / 66.10 / 54.61 / 29.98 / 18.04 / 177.32 / 179.25
L / 208 / L / 208 / L208 / 54.62 / 18.14 / 42.19 / 179.51 / 179.84
V / 209 / V209 / 57.94 / 64.58 / 179.91 / 178.10 / V / 209 / V209 / 57.94 / 64.58 / 179.91 / 178.10
P / 238 / P / 238
I / 239 / I / 239 / I239 / 64.73 / 62.69 / 33.30 / 43.12 / 179.31 / 177.58
A / 240 / A240 / 62.66 / 55.65 / 17.67 / 177.78 / 179.22 / A / 240 / A240 / 62.66 / 55.65 / 17.67 / 177.78 / 179.22
V / 288 / V288 / 60.44 / 67.20 / 30.03 / 31.21 / 177.55 / 177.94 / V / 288 / V288 / 60.44 / 67.20 / 30.03 / 31.21 / 177.55 / 177.94
R / 289 / R / 289 / R289 67.20 59.99 31.07 29.71 178.02 179.58
L290 59.89 57.90 29.87 40.93 179.63 179.46
F291 57.94 61.92 179.40 178.77
K292 61.94 61.61 39.45 31.82 178.83 179.07
Q293 61.52 59.67 179.14 179.41
L / 290 / A151 55.29 55.42 180.35 179.80
Y152 55.34 57.63 37.88 179.67 176.98
A153 57.77 55.86 177.06 178.71
E158 56.08 60.16 178.67 177.51
D159 60.12 58.29 177.62 178.45
L295 58.26 59.28 39.96 42.64 178.52 178.81
S296 59.27 61.96 42.59 63.18 178.91 177.19 / L / 290
F / 291 / F / 291
K / 292 / K / 292
Q / 293 / Q / 293
L / 294 / L / 294 / L294 / 179.41 / 178.54
L / 295 / L / 295 / L295 / 58.26 / 59.28 / 39.96 / 42.64 / 178.52 / 178.81
S / 296 / S / 296 / S296 / 59.27 / 61.96 / 42.59 / 63.18 / 178.91 / 177.19
S / 297 / S297 / 61.85 / 63.20 / 177.32 / S / 297 / S297 / 61.85 / 63.20 / 177.32
F / 298 / D162 / 59.28 / 57.63 / 39.33 / 180.91 / 177.76 / F / 298 / F298 / 62.48 / 62.66 / 177.40 / 177.70
Q / 299 / R163 / 57.45 / 57.26 / 29.92 / 177.74 / 176.30 / Q / 299 / Q299 / 62.56 / 59.24 / 39.63 / 28.30 / 177.66 / 179.19
R / 300 / R / 300 / R300 / 59.33 / 58.77 / 28.37 / 29.64 / 179.18 / 177.53
N / 301 / N301 / 58.78 / 54.44 / 41.79 / 177.55 / 175.21 / N / 301 / N301 / 58.78 / 54.44 / 41.79 / 177.55 / 175.21
Legend to Table S8.
The table highlights the 16 differences between EZ-ASSIGN (left column) and the hand assignment (right column) of the resonances of ATPase. Black entries show some of the 320 assignments that do correspond. The GSS are labeled according to the hand assignment.
Corresponding labels in the two columns have the same font color. Dark blue entries in the right column were unassigned by EZ-ASSIGN.
The differences are rationalized as follows.
The grey block R161-H166 in the hand-assignment has 2 or more rung matches for the tri peptide 162-164 only. In the EZ-ASSIGNMENT, residue D162 is assigned with GSS 293 , which is involved in a tetra peptide stretch of 2 or more rung matches which is superior. The latter GSSs (L290-Q293) were placed at residues 159-162 because residues L290-S296 (light green block) were already occupied by seven GSS all connected by two rungs or more. According to our protocol (longest stretches with the largest number of matches are placed first), this is also a superior assignment. However, NOE data supported the hand assignment data.