Structural studies of the T4-DNA helix-destabilizing protein GP32*I by three-dimensional electron microscopy and image analysis.

Abstract

The three-dimensional (3-D) structure of gp32*I, a major proteolytic fragment of the DNA helix-destabilizing protein from bacteriophage T4, has been determined at 18 A resolution by electron microscopy of negatively stained crystals and computer image analysis. The crystalline areas processed in 3-D have the symmetry of the space group P2₁, with a = 47 Å, b = 63 Å, c = 65 Å, and α = β = γ = 90°. This P2₁ unit cell contains one gp32*I molecule per asymmetric unit. The molecule is roughly V-shaped, containing two large domains linked by a smaller domain occupying the base of the V. The total length of the molecule is about 110 Å with an average diameter of about 25 Å. Systematic analysis of the symmetry in images of untilted crystals determined that the crystal could display several types of projection symmetry, pgg, pg corresponding to P2₁ symmetry with the screw axis along the a axis of the crystal, and pg corresponding to P2₁ symmetry with the screw axis along the b axis. Among images displaying pg symmetry along the b axis, two types of images with noticeably different appearances were obtained. A hypothesis was formed that explained the different types of symmetry as the result of the growth of the gp32*I crystal in the space group P2₁ 2₁ 2₁, in steps of 1/2 of a unit cell along the thin direction of the crystal. Two different types of 1/2 unit cell thick steps were postulated. Computer simulations were used to generate synthetic images of untilted crystals containing either one, two or three steps of each kind. The results of the simulations prove that the space group of the gp32*I crystal is P2₁ 2₁ 2₁. They suggest that careful analysis of the symmetry in images of untilted gp32*I crystals can provide information about the thickness of the crystals. A strategy is presented for determining the structure of the gp32*I crystal at higher resolution by electron microscopy of frozen, hydrated crystals. This strategy includes the use of symmetry analysis as a tool for determining the thickness of the crystals so that data from crystals of the same thickness can be combined in 3-D. A similar approach may prove useful in the 3-D electron microscopic analysis of other thin, multi-layered crystals.