Engel A, Baumeister W, Saxton W O
Proc Natl Acad Sci U S A. 1982 Jul;79(13):4050-4. doi: 10.1073/pnas.79.13.4050.
A mass map of the hexagonally packed intermediate layer (HPI-layer), a regular protein monolayer from the cell envelope of Micrococcus radiodurans, has been obtained by scanning transmission electron microscopy. Samples were freeze-dried within the microscope, and low-dose images were recorded in the dark-field mode directly in digital form and processed by correlation averaging. The averaged projection of the unstained structure--i.e., the mass map--thus calculated shows a resolution to 3-nm period and reveals morphological features consistent with those obtained by negative staining. The mass of individual morphological domains was extracted by using variously the mass map itself or an average from a negatively stained HPI layer to define the domain boundaries. Protrusions as small as 1,300 daltons could be measured reproducibly within the unit cell of 655,000 daltons. The method developed opens an avenue to identify molecular species in situ and to correlate topographic information with biochemical data.
通过扫描透射电子显微镜获得了来自耐辐射微球菌细胞包膜的规则蛋白质单层——六角密排中间层（HPI层）的质量图。样品在显微镜内进行冷冻干燥，低剂量图像以数字形式直接在暗场模式下记录，并通过相关平均法进行处理。由此计算出的未染色结构的平均投影——即质量图——显示出3纳米周期的分辨率，并揭示出与负染色获得的形态特征一致的形态特征。通过使用质量图本身或负染色HPI层的平均值来定义结构域边界，提取了各个形态结构域的质量。在655,000道尔顿的晶胞内，可以可重复地测量到小至1300道尔顿的突起。所开发的方法为原位鉴定分子种类以及将地形信息与生化数据相关联开辟了一条途径。
