Initial refinement by simulated annealing

The refinement is begun with rigid body refinement, which is important if the model has come from molecular replacement. The positions of the two molecules in the asymmetric unit is optimized using the CNS task file rigid.inp. In this example 2 cycles of 20 steps are performed to ensure convergence. This can be important if the model is a significant distance from the correct position.

      cns_solve < rigid.inp > rigid.out [3 minutes]

The result of the rigid body refinement is a new coordinate file (rigid.pdb). In the majority of CNS refinement task files information about the refinement procedure is written out at the top of the output coordinate file (as REMARK statements):

REMARK coordinates from rigid-body refinement
REMARK refinement resolution: 500 - 1.8 A
REMARK starting r= 0.4306 free_r= 0.4423
REMARK final    r= 0.4236 free_r= 0.4307
REMARK wa= 3.20912
REMARK target= mlf cycles= 2 steps= 20
REMARK sg= P2(1)2(1)2(1) a= 65.508 b= 72.216 c= 45.035 alpha= 90 beta= 90 gamma= 90
REMARK parameter file 1  : CNS_TOPPAR:protein_rep.param
REMARK molecular structure file: mbp.mtf
REMARK input coordinates: mbp.pdb
REMARK reflection file= mbp.cv
REMARK ncs= none
REMARK B-correction resolution: 6.0 - 1.8
REMARK initial B-factor correction applied to fobs :
REMARK   B11=  -4.190 B22=   2.445 B33=   1.745
REMARK   B12=   0.000 B13=   0.000 B23=   0.000
REMARK B-factor correction applied to coordinate array B:   -6.838
REMARK bulk solvent: density level= 0.382686 e/A^3, B-factor= 28.7073 A^2
REMARK reflections with |Fobs|/sigma_F < 0.0 rejected
REMARK reflections with |Fobs| > 1000 * rms(Fobs) rejected
REMARK anomalous diffraction data was input
REMARK theoretical total number of refl. in resol. range:     38245 ( 100.0 % )
REMARK number of unobserved reflections (no entry or |F|=0):    323 (   0.8 % )
REMARK number of reflections rejected:                            0 (   0.0 % )
REMARK total number of reflections used:                      37922 (  99.2 % )
REMARK number of reflections in working set:                  34248 (  89.5 % )
REMARK number of reflections in test set:                      3674 (   9.6 % )
CRYST1   65.508   72.216   45.035  90.00  90.00  90.00 P 21 21 21
REMARK FILENAME="/tmp_mnt/Net/franklin/u0/pvm/tmp/mbp_ref/rigid.pdb"
REMARK DATE:10-Jun-99  13:14:10       created by user: paul
REMARK VERSION:0.5
ATOM      1  CB  LYS A 109      16.069   7.290  46.989  1.00 13.16      A
ATOM      2  CG  LYS A 109      17.021   6.260  47.596  1.00 13.16      A

This information provides a summary of the refinement and also a record of the input data and parameters used to generate this structure.

Following rigid body refinement, simulated annealing using torsion angle dynamics is used to improve the model. The use of torsion angle dynamics reduces the number of parameters being refined and hence reduces the degree of overfitting of the data. For an initial model with relatively large errors (due to manual building or misplaced atoms) a starting temperature of 5000K is recommended. In order to decrease the computational time required the colling rate can be increased from 25K to 50K. The simulated annealing refinement task file includes energy minimization both before and after the simulated annealing. Multiple refinement trials can be performed, each with different initial velocities for the molecular dynamics. It can be useful to run 5 or 10 trials if the there are some serious errors in the model - greater variation is usually seen in these areas. The structure factors from the multiple models can also be averaged to reduce the noise in the electron density maps (see the CNS task file optimize_average.inp). The simulated annealing refinement is performed with the CNS task file anneal.inp:

      cns_solve < anneal.inp > anneal.out [45 minutes]

Note that because there is a dimer in the asymmetric unit we could use non-crystallographic symmetry as a restraint in the refinement (see the other tutorials). This has not been done here for the sake of clarity but should be used if possible, especially if the resolution of the data is limited (worse than 2.5A). For example, if the refinement is continued with an annealing job using tight NCS symmetry restraints for residues 120 through 220 both the free R-value and the gap between the free R-value and R-value are decreased:

We will not use the NCS information in this refinement example, but it is clearly something that should be considered when possible.

The initial model has the same B-factor for all atoms. In order to take account of the possible variation in B-factors throughout the model, group B-factors are then refined. Two B-factors are refined for each residue, one for the main-chain atoms and one for the side-chain atoms. The small number of parameters being optimized allows this to be performed even when the resolution of the data is low (even below 3A). The group B-factor refinement is performed with the CNS task file bgroup.inp:

      cns_solve < bgroup.inp > bgroup.out [2 minutes]

This first round of refinement has reduced the free R-value from 44% to 40%. The next step is to locate any major part of the model which is currently missing (see the next section).