Sass H J, Büldt G, Beckmann E, Zemlin F, van Heel M, Zeitler E, Rosenbusch J P, Dorset D L, Massalski A
Department of Physics/Biophysics, Freie Universität Berlin, F.R.G.
J Mol Biol. 1989 Sep 5;209(1):171-5. doi: 10.1016/0022-2836(89)90180-0.
Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.
孔蛋白是一种整合膜蛋白，可在大肠杆菌的外膜上形成通道。对经负染色的二维孔蛋白晶体进行的电子显微镜研究表明，每个孔蛋白三聚体有三个染色积累区，揭示了跨膜孔的位置。在本研究中，使用配备氦冷却超导物镜的低温电子显微镜上的低剂量技术，对嵌入葡萄糖中的重组孔蛋白晶格进行了研究。将样品温度保持在5 K以提高显微镜分辨率和样品稳定性。在这些条件下，我们首次获得了孔蛋白晶格的电子衍射图，分辨率达到3.2 Å，以及显示光学衍射的图像，分辨率达到4.9 Å。对数字化显微照片应用相关平均技术，我们能够将孔蛋白三聚体的投影图像重建到高达3.5 Å的分辨率。在最终的投影图中，结合了电子衍射的振幅和这些图像的相位。主要特征是一条高密度窄带（厚度约6 Å），勾勒出三聚体的外周。由于光谱数据显示该分子几乎完全由β-折叠结构组成，我们得出结论，这条带是一个几乎垂直于膜平面穿过膜的圆柱形β-折叠片层。另一个有趣的发现是位于三聚体中心的低密度区域（约70 Ų）。
