The Shake-and-Bake structure determination of triclinic lysozyme

Abstract

The crystal structure of triclinic lysozyme, comprised of 1,001 non-H protein atoms and approximately 200 bound water molecules, has been determined ab initio (using native data alone) by the "Shake-and-Bake" method by using the computer program SnB. This is the largest structure determined so far by the SnB program. Initial experiments, using default SnB parameters derived from studies of smaller molecules, were unsuccessful. In fact, such experiments produced electron density maps dominated by a single large peak. This problem was overcome by considering the choice of protocol used during the parameter-shift phase refinement. When each phase was subjected to a single shift of +/-157.5 degrees during each SnB cycle, an unusually high percentage of random trials (approximately 22%) yielded correct solutions within 750 cycles. This success rate is higher than that typically observed, even for much smaller structures.

Figure 1

Plots of the unconstrained (R_unc) (a) and the constrained (R_con) (b) minimal function vs. SnB cycle. i, trials with default parameters that resulted in a uranium map; ii, a stability test with the known atomic positions as the starting peaks; iii, a correct solution where R_unc and R_con exhibit similar behavior; and iv, a correct solution where R_unc and R_con exhibit different behavior.

Figure 2

A histogram of final constrained minimal function values based on 512 random trials, resulting in a bimodal distribution with 116 correct solutions.

Figure 3

(Left) Peaks obtained from SnB (black) superimposed onto the triclinic lysozyme model from the PDB. The Cα trace is color-coded according to the temperature factor of the main chain of the model, from 3.5 (blue) to 15.0 (red). (Right) Cα traces for the triclinic lysozyme model color-coded according to the real space fit using program O (0.9 blue, 0.15 red) for main chain atoms with the electron density (E-maps) from SnB. Some residue numbers are marked.

Figure 4

Electron density E-map calculated with E-values and phases obtained from SnB (contoured at 3σ) for an ordered part of the structure where SnB finds many peaks. Both the SnB peaks (in black) and the PDB model are superimposed.

Figure 5

Electron density E-maps calculated with E-values and phases obtained from SnB for (Left) a less ordered part of the structure (contoured at 2.25σ) where SnB picks few of the 350 peaks found during a solution and (Right) the same region when SnB has been used to extend the solution in (Left) by picking 700 peaks (contoured at 2.5σ). The SnB peaks are superimposed in black, and the PDB model is also superimposed.

Figure 6

Electron density E-maps calculated with E-values and phases obtained from snb (contoured at 3σ) for the region around two of the disulfide bridges. The snb peak positions are indicated by the black circles and the PDB model also is superimposed.