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Dipolar Couplings for Structure Refinement 309 Fig. 4. Example of the input format for residual dipolar coupling-derived restraints (7) in X-PLOR (23). Residue 500 OO, X, Y, Z correspond to the origin, x-, y-, and z-axes of the alignment tensor axis system, respectively. Restraints are made with reference to the N–HN bond vector for each residue. The value in the second column from the right is the measured residual dipolar coupling. The far right number (0.2000) corresponds to the experimental measurement error, however this is not taken into consideration in the simulated annealing protocol. Edipolar = kdipolar ( 1DNH,calc – 1DNH,obs) 2 (5) where kdipolar is a force constant, and 1DNH,calc and 1DNH,obs are calculated and observed values of the residual dipolar couplings. This force constant is scaled in tandem with the NOE force constant during the cooling phase of the simulated annealing protocol. The value for kdipolar is chosen empirically, such that the difference between the calculated vs experimental values for 1DNH approximates the measurement error. 3. If the number of measured 1DNH values is limited, as in the case of the LDLR-AB pair, estimates for Da and R may be refined using a grid search method (8). We have used this method to optimize the two parameters independently. The procedure involves calculating series of structure ensembles (20 structures per ensemble) with varying values for either Da and R. For the 10 lowest total energy structures for each ensemble, estimates for Da and R were evaluated based on
310 Boyd et al. total and NOE energies. D a was incremented from 22.5 to 25.0 Hz in steps of 0.5 Hz, and R was incremented from 0.12 to 0.20 in steps of 0.02. For the LDLR- AB pair, optimal agreement was found with D a 23–23.5 Hz and R 0.16–0.18. These values are consistent with the estimates obtained based on analysis of the histogram shown in Fig. 3. 4. Introduction of residual dipolar coupling-derived restraints may result in violation of a subset of the NOE restraints. This is caused by inaccuracies in the NOEderived distances because of spin diffusion, time averaging, or spectral overlap. In this case, the NOE restraint list may be refined iteratively against the residual dipolar coupling restraints, until the structures satisfy selection criteria. For the LDLR-AB pair, structures shown in Fig. 5 were chosen with no NOE restraint violated by more than 0.5 Å, no dihedral angle restraint violated by more than 5°, and | 1 D NH,calc – 1 D NH,exp| < 2 Hz. 3.6. Validation of Structure Refinement 1. Although chemical shifts are dependent on molecular alignment, changes in 13C’ chemical shifts (∆δ13C’), which are particular sensitive to this effect, have been used to evaluate protein structures before and after refinement against residual dipolar coupling-derived restraints via use of a quality (Q) factor defined as: Q = rms(∆δmeas – ∆δpred )/rms(∆δmeas ) (6) where ∆δ is the change in chemical shift observed when shifting from an isotropic to an aligned medium (28). In the absence of 13C chemical shift data for the LDLR-AB pair, we have chosen instead to evaluate the improvement in structures based on T1/T2 ratios, which were not incorporated into the structure refinement. The T1/T2 ratio depends on the angle that NH bond vector angle with respect to the the diffusion tensor (see Chapter 22 in this volume). Therefore a comparison of (T1/T2) calc – (T1/T2) meas , via analysis of χ2 values for the structural ensembles computed before and after refinement against the residual dipolar coupling-derived restraints, should directly probe changes in the accuracy of the structure determination. χ2 is defined as: χ 2 = i ∑ {[(T 1/T 2) meas – (T 1/T 2) calc ] i / err [(T 1/T 2) meas ] i} 2 (7) where i is summed over values for residues that do not manifest large amplitude internal motions or conformational exchange line broadening. For the LDLR-AB pair, this subset of residues was defined as those localized to β-strands excluding residues affected by either fast or slow motions ({ 1 H}– 15 N-NOE > 0.7 and T 2 > 90 ms). As expected, the LDLR-AB families of structures were best-fit using a prolate ellipsoid model with D // = D zz and D ⊥ = D xx = D yy and D //> D ⊥, where D is defined as the molecular rotational diffusion tensor. Twenty structures were selected from ensembles calculated before and after residual dipolar coupling based refinement based on total energies and agreement with experimental constraints. The average χ 2 for each family dropped from and average value of 120 to 109 upon refinement, an approx 9% improvement.
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Methods in Molecular BiologyTM Biol
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M E T H O D S I N M O L E C U L A R
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© 2002 Humana Press Inc. 999 River
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Preface Calcium plays an important
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Contents Dedication ...............
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Contents xi 26 Enzymatic Assays to
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xiv Contents of Companion Volume 15
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xvi Contributors LESLIE D. HICKS
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20 Dean, Kelsey, and Re
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2 Yazawa
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4 Yazawa Fig. 1. Schematic represen
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6 Yazawa Fig. 2. Shop drawing for t
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8 Yazawa DuPont-NEN and the molar c
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10 Yazawa Fig. 4. Examples of the C
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12 Yazawa 5. Plot the calculated Ca
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14 Yazawa 12. Starovasnik, M. A., D
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16 Fig. 1. Molecular structures and
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18 Linse 7. 0.1 M EDTA. Dissolve 37
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20 Linse iterate the other paramete
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22 Linse tive to small alterations
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24 Linse References 1. Tsien, R. Y.
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26 Haiech and Kilhoffer to use the
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28 Haiech and Kilhoffer where γ is
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30 Haiech and Kilhoffer reporter gr
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32 Haiech and Kilhoffer tein with i
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34 Haiech and Kilhoffer Calmodulin
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36 Haiech and Kilhoffer Fig. 3. Mec
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38 Haiech and Kilhoffer We may sugg
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40 Haiech and Kilhoffer 29. Stewart
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42 Haiech and Kilhoffer 62. Becking
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44 Martin and Bayley Fig. 1. Absorp
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46 Martin and Bayley evaporation. C
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48 Martin and Bayley 4. Set the tem
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50 Martin and Bayley Fig. 2. Near-U
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52 Martin and Bayley discussed by M
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54 Martin and Bayley References 1.
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20 Dean, Kelsey, and Reik
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58 Fabian and Vogel are equipped wi
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60 Fabian and Vogel The penetration
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62 Fabian and Vogel perature, ionic
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64 Fabian and Vogel Fig. 3. IR spec
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66 Fabian and Vogel Fig. 4. Lower t
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68 Fabian and Vogel These correlati
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70 Fabian and Vogel Fig. 5. Amide I
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72 Fabian and Vogel 4. ATR-FTIR spe
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74 Fabian and Vogel 25. Georg, H.,
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76 Weljie and Vogel Fig. 1. Simplif
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78 Weljie and Vogel Fig. 3. Steady-
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80 Weljie and Vogel VIS spectrophot
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82 Weljie and Vogel 4. A series of
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84 Table 1 Advanced Fluorescence Me
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86 Weljie and Vogel References 1. L
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90 Johnson and Tikunova is removed
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92 Johnson and Tikunova function of
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94 Johnson and Tikunova Fig. 2. Rat
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96 Johnson and Tikunova Fig. 3. Rat
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98 Johnson and Tikunova Fig. 4. Ca
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100 Johnson and Tikunova cence inte
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102 Johnson and Tikunova 6. Johnson
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104 Julenius Fig. 1. Under conditio
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106 Julenius 3. Mix 50 µL of NHS s
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108 Julenius Fig. 3. A typical sens
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110 Julenius (2), is used in the Ia
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114 Lopez and Makhatadze Fig. 1. Th
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116 Lopez and Makhatadze 6. Buffers
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118 Lopez and Makhatadze The excess
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122 Lopez and Makhatadze Fig. 1. Is
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124 Lopez and Makhatadze 2.5 mL are
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126 Lopez and Makhatadze pendence o
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128 Hicks et al. equipped with a pu
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130 Hicks et al. Fig. 2. Debye plot
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132 Hicks et al. and 1.0 mg/mL for
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134 Hicks et al. Fig. 3. Sedimentat
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136 Hicks et al. 5. Hayes, D. B. (M
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138 Trewhella and Krueger This chap
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140 Trewhella and Krueger of the sc
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142 Trewhella and Krueger Table 1 C
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144 Trewhella and Krueger beam, or
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146 Trewhella and Krueger At very l
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148 Trewhella and Krueger analytica
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150 Trewhella and Krueger collapsed
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152 Trewhella and Krueger P(r) func
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154 Trewhella and Krueger Fig. 3. S
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156 Trewhella and Krueger concentra
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158 Trewhella and Krueger by a Nati
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162 Doherty-Kirby and Lajoie metal
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164 Doherty-Kirby and Lajoie two Ca
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166 Doherty-Kirby and Lajoie 7. Pep
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168 Doherty-Kirby and Lajoie Fig. 1
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170 Doherty-Kirby and Lajoie Fig. 2
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172 Doherty-Kirby and Lajoie 5. Alt
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174 Doherty-Kirby and Lajoie phosph
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176 Shaw Fig. 1. Ribbon drawing of
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178 Shaw 2.3. Other 1. Chemicals fo
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180 Shaw 3.3.3. Purification 1. Con
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182 Shaw 10. Hodges, R. S., Semchuk
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184 Brokx and Vogel Table 1 An Over
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186 Brokx and Vogel Fig. 1. UV abso
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188 Brokx and Vogel Fig. 2. 20% SDS
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190 Brokx and Vogel it through an a
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192 Brokx and Vogel 8. Weljie, A. M
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196 Berliner Fig. 1. Aqueous X-band
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198 Berliner in the Bruker instrume
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200 Berliner Fig. 3. ESR spectra of
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202 Berliner Fig. 5. Low-temperatur
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204 Berliner 14. Musci, G., Reed, G
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206 Clarke and Vogel cal calcium-bi
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208 Clarke and Vogel Fig. 1. 113 Cd
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214 Clarke and Vogel The linewidth
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218 Drakenberg sands, of Hertz broa
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220 Drakenberg 1. Bloch equations m
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222 Drakenberg Fig. 2. Measurement
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224 Drakenberg Fig. 3. (A) 43 Ca NM
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226 Drakenberg Fig. 5. 43 Ca NMR sp
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228 Drakenberg NMR at sub-mM concen
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230 Drakenberg 30. Shimizu, T., Hat
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232 Weljie and Heringa Table 1 Amin
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234 Weljie and Heringa Table 2 Webs
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236 Weljie and Heringa diction meth
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238 Weljie and Heringa these databa
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240 Weljie and Heringa lineages. Th
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242 Weljie and Heringa compared, an
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244 Weljie and Heringa sequences as
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246 Weljie and Heringa much larger
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248 Weljie and Heringa ment require
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250 Weljie and Heringa 10. Kawasaki
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252 Weljie and Heringa 44. Thompson
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254 Weljie and Heringa
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256 Li et al. In order for these ex
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Page 281 and 282: 262 Li et al. sion system works eff
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Page 287 and 288: 268 Mal et al. NMR data, X-PLOR (11
Page 289 and 290: 270 Mal et al. Table 1 Simulated An
Page 291 and 292: 272 Mal et al. 3.1.2.1. AMBIGUOUS D
Page 293 and 294: 274 Mal et al. molecular dynamics a
Page 295 and 296: 276 Mal et al. Assuming a rigid bod
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Page 299 and 300: 280 Mal et al. References 1. Drenth
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Page 305 and 306: 286 Werner et al. Our research has
Page 307 and 308: 288 Fig. 1. (A) T 1, T 2 and the he
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Page 317 and 318: 298 Werner et al. 9. Reinhardt, D.
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Page 321 and 322: 302 Boyd et al. utilize residual di
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Page 337 and 338: 318 Yap et al. image representation
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Page 341 and 342: 322 Table 1 Angle and Distance Outp
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Page 345 and 346: 326 Kobayashi that S-100A1 and S-10
Page 347 and 348: 328 Kobayashi 3.2. Coupling of Crom
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Page 351 and 352: 332 Kobayashi 0.1% TFA at a flow ra
Page 353 and 354: 334 Kobayashi Both recombinant S-10
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Page 361 and 362: 342 Walsh et al. 11. Bovine serum a
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Page 367 and 368: 348 Walsh et al. 3.5. NOS Reduction
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Page 375 and 376: 356 Hughes et al. The electroporati
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360 Hughes et al. range, repeat ste
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362 Hughes et al. Table 1 Examples
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366 Persechini of the different CaM
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368 Persechini Fig. 1. Schematic re
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370 Persechini 2. 1 L of Terrific b
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372 Persechini Fig. 4. Titration wi
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374 Persechini Table 1 Parameters f
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376 Persechini Fig. 6. The [Ca 2+ -
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378 Persechini determined if the di
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380 Persechini relatively insensiti
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382 Persechini 18. Chafouleas, J. G
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384 Török et al. actions in the c
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386 Török et al. 3. The reaction
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388 Török et al. Fig. 2. Electros
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390 Török et al. Fig. 3. Peptides
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392 Török et al. Table 1 Peptide
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394 Török et al. Fig. 7. Nanospra
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396 Török et al. Fig. 9. Nanospra
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398 Török et al. Fig. 11. Electro
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400 Török et al. 3 µM FL-calmodu
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402 Török et al. Fig. 14. Localiz
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404 Török et al. Fig. 16. Indicat
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406 Török et al. the significantl
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408 Török et al.
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410 Index substitutes, see Fluoresc
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412 Index Stern-Volmer plot, 78, 81
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414 Index Phenothiazines, see Calmo
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METHODS IN MOLECULAR BIOLOGY TM •
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