Protein Engineering vol.7 no.2 pp.
235-241, 1994
A study of simulated annealing protocols for use with molecular
dynamics in protein structure prediction
C.A.Laughton
CRC Biomolecular Structure Unit, Institute of Cancer Research, Sutton,
Surrey SM2 5NG, UK
The method of simulated annealing can be of use in protein
structure prediction by homology modelling where side chain
conformations must be predicted. In this study an attempt
has been made to optimize a molecular dynamics method for
this purpose. Heating and cooling protocols to maximize the
accuracy of the predictions have been developed. The
optimized protocol involves cooling from 3000 to 0 K over
20 ps while simultaneously introducing the non-bonded
energy term. The use of a 'soft' non-bonded interaction
energy term in place of a standard 6 - 1 2 potential is found
to be important. The reliability of the predictions has been
analysed in terms of the environment of the residues (solvent
accessibility) and the degree of uncertainty in the structure
(number of unknown torsion angles). Depending on these
factors the percentage of unknown side chain torsion angles
that are correctly predicted within 30° ranges from ~ 50 to
75%. Potential problems and limitations of the method are
discussed.
Key words: modelling/molecular dynamics/protein side chain
conformation/protein structure prediction/simulated annealing
Introduction
The prediction of protein structures by homology modelling is
based on the observation that proteins showing reasonable
sequence homology and related function usually have very similar
3-D structures. Thus, if the structure of one of such a group of
proteins is determined, the structure of a related protein may be
predicted from it (Blundell et al., 1987; Greer, 1991). The
process of homology modelling generally involves three phases.
In the first phase information about the backbone coordinates of
the known structure and sequence alignments aid the prediction
of the overall tertiary fold of the homologous protein. In the
second phase portions of the main chain that could not be built
directly from the known structure Qoops) are predicted. In the
third phase the conformations of the amino acid side chains are
predicted. Methods of predicting side chain conformations can
be divided into two broad categories, though the distinction is
not absolute: (i) conformational search-based methods and
(ii) rule-based methods.
The first approach involves attempting to find the global energy
minimum in the n-D torsional conformational space defining the
orientation of all side chains by some form of searching. To
perform such a search in an exhaustive manner takes a time
propotional to r", where r is the number of values of each
torsion angle that is examined; this rapidly becomes impracticable
as n increases. A number of approaches have been investigated
to help overcome this combinatorial problem (Desmet et al.,
1992). The search may be performed one residue at a time (Snow
and Amzel, 1986; Bruccoleri and Karplus, 1987; Schiffer et al.,
1990) or simultaneously only for residues within a local site. The
© Oxford University Press
dimensionality of the problem can be further reduced by
restricting the search to common rotamers for each side chain
(Tuffery et al., 1991; Wilson et al., 1993). Other methods replace
the exhaustive search with simulated annealing (Lee and Subbiah,
1991) and may include the rotamer approximation as well (Holm
and Sander, 1992a,b).
The alternative, rule-based methods of side chain prediction
use information about the side chain conformations in the known
structure to aid the prediction of the conformation of homologous
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residues in the protein being built and, thus, may be regarded
as extending the rationale behind phase 1 of the homology
modelling procedure (Sutcliffe et al., 1987; Summers and
Karplus, 1989). For example, if a methionine residue in the
known structure is replaced by an isoleucine residue in the
homologue, then the first two of the three side chain dihedral
angles for the methionine may be transferred to their equivalents
for isoleucine, thus providing a complete prediction of the
conformation of this residue. However, it will be obvious that
if the situation is reversed, the two torsion angles in an isoleucine
are insufficient to fully define the conformation of a replacement
methionine side chain. The mediod, then, in general provides
only a partial prediction of the side chain geometry and no
prediction at all in loop regions. For this reason the rule-based
methods need to be supplemented by conformational search-based
methods to provide a complete prediction. This does not mean
that the rule-based methods are not worth employing; they are
quick to apply and in general leave only a small fraction of the
dihedrals to be determined by searching. This reduction in the
dimensionality of the problem makes its solution more rapid. To
date, the use of simulated annealing methods in this context—
as an adjunct to rule-based procedures—has not been investigated
in detail.
Simulated annealing involves heating the molecule to such a
temperature that it has potential access to all the conformations
of interest, being able to overcome any large energy barriers that
may exist between them. The molecule is then slowly cooled so
that very gradually the highest energy conformations become
inaccessible, but escape from them to lower energy conforma-
tions remains possible. Ideally, as the temperature approaches
zero the molecule is trapped in the global minimum energy
conformation and if the force field is accurate, this should be
close to the true conformation. This procedure may be
implemented either through a Monte Carlo (MC) or molecular
dynamics (MD) simulation.
In either case, the success of the method depends critically on
the annealing protocol and the quality of the force field. There
has been some work on optimizing the MC approach in the field
of homology modelling (Chou and Carlacci, 1991; Lee and
Subbiah, 1991; Abagyan and Argos, 1992; Holm and Sander,
1992a,b) and the MD approach in the field of X-ray structure
refinement (Brunger and Krukowski, 1990). So far there has been
no systematic study of the MD protocol appropriate for homology
modelling. This paper presents the results of an investigation of
some of the potential variables, in particular heating/cooling
protocols and the treatment of electrostatic and non-bonded
235
C.A.Laughton

interactions. In addition, it analyses how the success of the method

can depend on factors outside the control of the investigator, in
particular the percentage of the torsion angles defining the
conformation of the molecule which are being determined by this
method and the solvent accessibility of the residues involved. For
this study a hypothetical homology modelling case has been used,
the prediction of the structure of cytochrome P450cam, based on
its own crystal structure (Poulos et al., 1987). This protein was
chosen because homology modelling based on P450C,, is an
area of much interest (Ferenczy and Morris, 1989; Laughton
et al., 1990; Graham-Lorence et al., 1991; Poulos, 1991;
Zvelebil et al., 1991; Lewis and Moereels, 1992; Zhou et al.,
1992). Although the approach has been developed here chiefly
as an adjunct to rule-based methods, its application to the
prediction of all the side chains in bovine pancreatic trypsin
inhibitor (BPTI) is also presented, as this enables the method to
be compared directly with others.

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Methodology
The crystal structures of cytochrome P450cam (Poulos et al.,
1987) and BPTI (Wlodawer et al., 1984) were obtained from
i... i.Ill illll.L. I. i . i .
the Brookhaven Protein Databank (Bernstein et al., 1977; Abola
Atortlon (")
et al., 1987). All molecular dynamics calculations were
performed using the AMBER 4.0 suite of programs (Weiner
et aL, 1986; Pearlman et al., 1991) on a Hewlett-Packard HP720
computer. The AMBER united-atom force field was used to
parameterize the structures. As the haem and camphor residues
in P450cam were held frozen during all calculations, no attempt
was made to assign realistic bond, angle or dihedral parameters
to these groups, however they were assigned appropriate partial
charges and non-bonded parameters. Solvent accessibilities of
the residues were determined using the method of Connolly
(1985). Residues described as 'buried' had less than half the
solvent-accessible surface area of the same residue in the sequence
G l y - X - G l y in an extended conformation.
To simulate a homology modelling situation for P450cam, a
Fig. 1. Distribution of torsion angles relative to initial values after 10 ps
percentage of the side chain dihedral angles were chosen at molecular dynamics at 3000 K (a) using a 6-12 van der Waals term scaled
random as 'undefined', as though application of a rule-based by 10~7. (b) using a 'soft' repulsion term scaled by 10~7, (c) as (b) but
method to a hypothetical homologous P450 had left them with initial velocities assigned at the final temperature.
unpredicted. Typically this method resulted in a selection of
residues of which 30% had one undefined torsion, 40% two, 20%
three and 10% four. All side chain atoms whose positions were up this space are dominated by non-bonded interactions, so
dependent on these torsion angles were then included in the transitions over them may be facilitated by raising the temperature
simulated annealing protocol, using the AMBER 'belly' option of the system and/or by reducing their height by scaling the
(i.e. all other atoms were 'frozen'). The protocol consisted of non-bonded interaction energy term. A combination of both
an initial minimization (to an r.m.s. gradient of <0.1 kcal/ approaches was used. Taking a random 10% of the side chain
mol/A), the simulated annealing run and then a final minimiza- torsions as flexible (torsion set 1) and scaling the electrostatic
tion. A distance-dependent dielectric constant (e = r,j) was used and van der Waals interactions by a factor of 10~7, 10 ps of
and a non-bonded cut-off of 9 A. Molecular dynamics simula- dynamics was performed at a range of temperatures, controlled
tions were performed with a time step of 1 fs and without Shake by coupling to a heat bath. The resulting structures were analysed
(in early experiments a time step of 2 fs was tried in association in terms of the deviations of the selected torsion angles from their
with Shake, but MD runs often terminated prematurely due to initial values. It was observed that, even at 3000 K, after 10 ps
failure of the algorithm under the annealing conditions). most torsion angles remained close to their original values (Figure
la). The situation was improved (Figure lb) when the standard
In the calculation of differences between torsion angles in AMBER van der Waals 6-12/10-12 potential was replaced by
different structures, allowance was made for symmetry for Asp, the alternative, 'soft', repulsion only potential:
Glu, Phe and Tyr residues.
E = K - r2)2 r < ro
Results
E = 0 r > rn
Heating protocols
The first stage in a simulated annealing procedure is to bring where r0 is the sum of the van der Waals radii of the two atoms
the system to a state in which it is able to explore all potentially concerned and K^ is a force constant, set equal to 1 kcal/
relevant parts of conformational space. The barriers which divide mol/A4 in this case. This form of potential has the advantage

236
 Prediction of protein side chain conformations

TaWe I. The effect of differing treatments of non-bonded interactions on the

accuracy of final annealed structures

_ so- Cooling K Electrostatics Af*1 Accuracy (all residues/

run" (kcal/mol/A4) (kcal/mol) buried only)
% torsionsc R.m.s.d."

1 1.0 off 101 68/74 0.61/0.52

2 1.0 off 95 68/70 0.67/0.43
3 1.0 off 175 64/63 0.58/0.41
4 1.0 on 56 59/63 0.60/0.49
5 1.0 on 54 68/70 0.60/0.43
6 1.0 on 29 65/72 0.56/0.40
7 0.2 off 66 71/77 0.52/0.39
8 0.2 off 46 65/72 0.55/0.39
tlm* (pt) 9 0.2 off 107 59/65 0.65/0.52
10 0.2 off 28 72/84 0.48/0.37
Fig. 2. Torsion angle randomization as a function of time and the 11 0.2 off -30 65/72 0.53/0.47
temperature of the molecular dynamics simulation. 12 0.2 off 72 78/91 0.49/0.33

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"For a full description of the protocols, see text.
b
Energy difference from minimized crystal structure.
^Percentage of dihedrals within ±30° of the crystal structure values.
d
Side chain atom r.m.s. deviation from crystal structure.

Ant (pi)
Fig. 3. Sampling of conformational space during a 10 ps molecular
dynamics simulation at 3000 K. Fractions of torsions that have sampled one,
two or all three conformational domains (r, g+, g~), as a function of time.
that its value remains finite even when the atomic separation is
zero. The ability of parts of the structure to 'pass through' other
parts without excessive hindrance is vital if all areas of conforma-
tional space are to be sampled. However, to obtain a satisfact-
ory spread in the torsion angles, it was also necessary to assign
the initial velocities in the system from a Boltzman distribution
at the required temperature, rather than rely on the coupling to
the heat bath to raise the temperature of the system from zero,
as was the case before (Figure lc).
Taking this last protocol, the effect of the duration and
temperature of the heating phase was examined. Runs of 10 ps
were performed at 1000, 2000 and 3000 K. Plotting the mean
absolute change in the torsions from their initial values as a
function of time (Figure 2), we see that even at 1000 K, most
of the randomization takes place in the first 2 ps, but that a full
10 ps at 3000 K probably gives the best chance of a well-
randomized and highly mobile system. To further demonstrate
the mobility of the system at 3000 K, the fraction of side chain
torsions that have experienced one, two or all three of the basic
conformational domains (t, g+ and g~) is plotted as a function
of time (Figure 3). By 2 ps some 60% of the torsions have already
been observed in all three domains at some stage, but even after
10 ps there are some 10% of the torsions that have remained
in their original domain and another 10% that have been observed
in only one other.
Cooling protocols
The final structure obtained using the last heating protocol
described above was used as the initial structure to test cooling
protocols. Each procedure was repeated three times with different
initial seeds for the random number generator which controls the
assignment of the initial velocities. At the end of each run, the
final structure was energy minimized with conventional and
unsealed non-bonded energy terms. The results were analysed
in terms of the final energy of the structures, compared to that
of the similarly minimized crystal structure and the percentage
of the flexible torsion angles that lay within 30° of their correct
values.
In the first protocol (cooling runs 1 - 3 ) , initial velocities were
assigned at 3000 K and the heat bath cooled from 3000 to 0 K
linearly over 50 ps, while the 'soft' non-bonded interactions were
scaled up linearly from a K^ of 10"7 to 1 kcal/mol/A4. The
electrostatic interactions remained scaled by 10~7. The second
protocol (cooling runs 4 - 6 ) was the same as the first, except
that the electrostatic interactions were also scaled up to full
strength over the last 40 ps of the simulation. The third protocol
(cooling runs 7-9) was also the same as the first, except that
the final value of K^ was reduced to 0.2 kcal/mol/A4. The
results are shown in Table I and the progress of the annealing
through the cooling is shown in Figure 4, where, for each
protocol, the mean error in the torsion angles is plotted as a
function of time.
The first protocol gave final structures with energies 9 5 -
175 kcal/mol above the minimized crystal structure and with
64-68% of the torsions correctly predicted. Considering just
'buried' residues, the predictive accuracy was 63-74%.
Introducing the electrostatic term led to the structures of better
energy, 29-57 kcaiymol above the target value, but the side chain
conformations were no better predicted. On examining the cooling
profiles (Figure 4), it was clear that most of the torsion angle
error correction occurred in the first 10 ps of the simulation,
during which time the value of K^ rose to 0.2 kcal/mol/A4. In
the third protocol, K^ reached this value only at the end of the
simulation, allowing more time for the structure to escape from
unfavourable conformations. This resulted in marginally better
performance and this protocol for the treatment of non-bonded

237
C.A.Laughton

Table D. The effect of the rate of cooling on the accuracy of final annealed
structures

Cooling Cooling Torsion Accuracy (all residues/

run1 time set (kcal/mol) buried only)
(ps) % torsions' R.m.s.d.d

13 5 1 102 55/53 0.57/0.48

14 5 1 141 55/60 0.64/0.56
15 5 1 139 59/63 0.58/0.55
16 5 2 182 61/70 0.68/0.71
17 5 2 94 60/74 0.51/0.45
18 5 2 42 64/77 0.53/0.48
19 10 1 109 74/74 0.42/0.37
20 10 1 98 64/70 0.57/0.39
21 10 1 85 58/67 0.52/0.43
22 10 2 39 68/77 0.48/0.45
23 10 2 159 60/60 0.56/0.56
24 10 2 67 68/84 0.51/0.45

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25 20 1 93 62/63 0.60/0.41
26 20 1 126 67/70 0.65/0.43
27 20 1 97 64/70 0.59/0.56
28 20 2 102 76/84 0.56/0.44
29 20 2 49 63/72 0.60/0.47
30 20 2 98 69/84 0.48/0.42
31 20 3 91 72775 0.53/0.53
32 20 3 42 57/69 0.74/0.44
33 20 3 101 61/69 0.56/0.47

"For a full description of the protocols, see text.

•"Energy difference from minimized crystal structure.
Percentage of dihedrals within ±30° of the crystal structure values.
d
Side chain atom r.m.s. deviation from crystal structure.

Fig. 4. Torsion angle optimization during 50 ps simulated annealing cooling

runs with the temperature falling linearly from 3000 to 0 K and (a) K^
increasing linearly from 10~7 to 1 kcal/mol/A4, (b) as (a) but with the
electrostatic term being scaled from 10~7 to 1 over the last 40 ps and (c) as
(a) but to a final value of K^ of 0.2 kcal/mol/A4.

interactions was settled on. The protocol was repeated using a

different random 10% of torsions (torsion set 2, runs 10—12).
This set gave rather better results, final energy differences ranging
from - 3 0 to 72 kcal/mol and up to 78% of all torsions and 91 %
of buried ones being correctly predicted.
A total cooling period of 50 ps was chosen originally as being
the longest that was practicable. Attempts were now made to see
by how much this could be shortened without affecting the
accuracy of the method. Both sets of 10% of the torsions were
investigated and as before each cooling run was repeated three
times with different random number seeds. The results are shown
in Table II (cooling runs 13—30) and the averaged results are
plotted in Figure 5. In all cases, the second set of torsions gave
slightly better results than the first. In general, the mean accuracy
of the predictions appeared to increase with the cooling time,
but the improvement from 20 to 50 ps was very slight. In contrast,
a major improvement in the mean energy of the final structures
was observed between 20 and 50 ps. Since it is accuracy, rather
than energy, which is the goal, the 20 ps cooling period was
selected as the optimum. A third random 10% of torsion angles
was selected (torsion set 3) and put through the 20 ps protocol
to further check the variability in the accuracy of the prediction
(cooling runs 31-33). It gave results very similar to those for
the first random 10% of torsions (Table H).
The next part of the study investigated the dependence of the
• — Mt 1 (al)
• Mt2(U)
--••-- MI 1 (buned)
------ MI 2 (b»«d)
SO
Cooling tlm* (pi)
Fig. 5. Effect of cooling time (from 3000 K) on the accuracy of the
annealed structures (percentage of torsion angles within 30° of the crystal
structure values). Results for two different random sets of torsions are
shown. All values are the means of three separate runs with different initial
velocity distributions.
accuracy of the method on the percentage of the torsions which
were to be predicted. Six different random selections of the side
chain torsions were made, three of 20% (torsion sets 4 - 6 ) and
three of 30% (sets 7-9). As before, after the heating phase each
20 ps cooling run was repeated three times (cooling runs 34-51).
The results are shown in Table m . For the three sets of 10%
of the torsions, the mean accuracy of the annealed structures was
66% taken over allflexibletorsions or 73% considering just those
belonging to buried residues. At 20% of the torsions these values
were 63 and 67% respectively and at 30% they were 63 and 70%.

238
 Prediction of protein side chain conformations

Table in., The effect of the number of flexible torsions on the accuracy of Table VI. Application of the method to BPTI. Side chain atom r.m.s.
final annealed structures deviations and solvent accessibilities for individual residues from each
annealing run
Cooling Torsion Fraction of Accuracy (all residues/
run* set dihedrals (%) (kcal/mol) buried only) Residue Solvent Side chain r.m.s . deviation (A)
c
R.m.s.d." exposure A B C D E Mean'
% torsions

34 4 20 194 63/67 0.91/0.57 1.053 1.109 0.987 1.046 1.124 0.961

Arg 1 79
35 4 20 385 63/67 0.95/0.65 0.138 0.135 0.138 0.132 0.140 0.140
Pro 2 50
36 4 20 162 64/67 0.77/0.60 0.348 0.337 0.354 0.861 0.349 0.366
Asp 3 89
37 5 20 129 66/70 0.86/0.70 1.453 1.422 1.446 1.420 0.248 1.350
Phe 4 46
38 5 20 212 64/67 0.86/0.84 5 0.000 0.000 0.000 0.000 0.000 0.000
Cys 5
39 5 20 246 60/68 0.80/0.58 0.257 0.230 0.830 0.809 0.837 0.817
Leu 6 82
40 6 20 179 64/70 0.93/0.73 1.236 1.255 1.248 1.235 1.240 1.247
Glu 7 73
41 6 20 317 61/63 0.94/0.72 0.184 0.187 0.131 0.182 0.182 0.175
Pro 8 88
42 6 20 188 59/64 0.93/0.66 0.057 0.107 0.200 0.046 0.064 0.063
Pro 9 57
43 7 30 340 62/71 1.09/0.93 10 0.291 1.158 0.129 0.409 0.290 0.695
Tyr 61
44 7 30 249 67/75 0.87/0.70 0.095 0.145 0.121 0.990 0.584 0.148
Thr 11 58
45 7 30 243 63/70 1.00/0.76 0.378 0.380 0.402 0.372 0.383 0.371
Pro 13 71
46 8 30 330 59/66 0.99/0.75 0.000 0.000 0.000 0.000 0.000 0.000

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Cys 14 52
47 8 30 229 60/75 1.01/0.73 0.508 0.453 0.508 0.501 0.536 0.470
Lys 15 98
48 8 30 313 59/68 0.98/0.72 1.634 1.440 0.811 1.360 0.848 1.411
Arg 17 85
49 9 30 380 60/65 1.00/1.00 18 0.735 1.788 0.108 1.873 0.972 0.974
De 49
50 9 30 584 66/67 1.08/0.86 19 74 0.734 0.358 0.345 0.741 0.749 0.738
lie
51 9 30 311 67/72 0.92/0.74 1.133 1.276 0.855 0.737 1.391 0.777
Arg 20 34
0.050 0.075 0.112 0.068 0.072 0.105
Tyr 21 38
'For a full description of the protocols, see text. 0.181 0.196 1.168 0.186 0.187 0.149
b Phe 22 25
Energy difference from minimized crystal structure. 0.124 0.610 0.084 0.152 0.170 0.210
'Percentage of dihedrals within ±30° of the crystal structure values. Tyr 23 26
1.463 0.266 0.893 0.281 1.450 0.481
d
Side chain atom r.m.s. deviation from crystal structure. Asn 24 39
0.832 1.076 1.055 0.492 1.046 1.061
Lys 26 97
0.172 0.140 0.887 1.169 1.155 0.149
Leu 29 70
0.000 0.000 0.000 0.000 0.000 0.000
Cys 30 39
Table IV. The effect of the number of flexible (undetermined) dihedrals in 0.645 1.583 1.151 1.129 0.773 0.685
Gin 31 53
a residue on the accuracy of its predicted structure 0.095 0.351 0.356 0.092 0.563 0.095
Thr 32 69
0.899 1.068 0.917 0.943 0.867 0.778
Cooling % accuracy1' Phe 33 28
0.684 0.644 0.036 0.449 0.645 0.659
run* Val 34 52
One Two Three Four 2.208 0.075 0.053 2.178 0.078 0.168
torsion torsions torsions torsions Tyr 35 15
0.926 0.953 0.906 0.955 0.929 0.920
Cys 38 35
0.936 0.945 1.460 0.794 0.929 0.823
34 74 60 56 45 Arg 39 82
0.690 0.874 1.400 1.049 0.659 0.778
35 68 71 58 60 Lys 41 87 1.237
1.092 1.529 1.383 1.401 1.251
36 74 61 ^ 58 40 Arg 42 82 0.342 0.874 0.270 0.167 0.347 0.063
Asn 43 30 0.964 0.387 0.399 0.857 0.753 0.236
•For a full description of the protocols, see text. Asn 44 48 0.114
0.138 0.178 0.142 0.209 0.104
"Tn terms of the percentage of dihedrals within ±30° of the crystal Phe 45 26 0.336 0.271 0.893 1.153 0.756 0.835
structure values. Lys 46 97 0.356 0.453 0.329 0.486 0.488 0.460
Ser 47 51 1.253 1.268 1.280 1.254 0.480 1.246
Glu 49 74 0.446 0.472 0.478 0.473 0.469 0.422
Table V. Application of the method to the prediction of all the side chain Asp 50 59
dihedral angles in BPTI 0.078 0.110 0.078 0.000 0.096 0.051
Cys 51 0 1.278 1.089 1.137 1.297 1.080 1.030
Annealing run % accuracy* All-atom Side chain £* Met 52 71 1.097 1.062 0.977 1.431 1.411 0.976
r.m.s.d. r.m.s.d. (kcal/mol) Arg 53 87 0.112 1.409 0.121 0.130 0.126 0.123
(A) (A) Thr 54 59 0.000 0.000 0.000 0.000 0.000 0.000
Cys 55 5
A 56(68) 1.6 (1.8) 2.3 (2.4) -228
B 48 (58) 2 .0 (2.4) 2.9 (3.2) -193 •Coordinate averaged and reminimized structure.
C 51 (65) 1.6 (1.7) 2.3 (2.4) -217
D 54(71) 1.7 (1.7) 2.4 (2.3) -216
E 57(71) 1.3 (1.1) 1.9(1.6) -232 residues having just one torsion angle to be predicted by simulated
Mean 53(67) 1.7 (1.7) 2.3 (2.4) annealing, the success rate was 72%. This fell to 64% where
Averaged and reminimized 55(71) 1.4 (1.3) 2.0 (1.7) -277 two angles had to be determined, 57% for three and 48% for four.
Application of the method to BPTI
•In terms of the fraction of dihedrals within ± 30° of the crystal structure
values. Although the method has been developed as an adjunct to rule-
^Molecular mechanics energy. based procedures for side chain prediction, it was applied to the
Figures in parentheses refer to measurements over buried residues only. prediction of all the side chains of BPTI, as such a test has
commonly been employed in other studies and so it enables a
For one 30% set of torsions (set 7), the accuracy of the predictions more detailed evaluation of the method to be made. Except that
(cooling runs 43-45) was analysed in terms of the number of all the torsion angles (with the exception of those from cross-
flexible torsions in the residues concerned (Table IV). For linked cysteines) were included in the simulation, the protocol

239