Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 angstrom resolution by electron cryomicroscopy and angular reconstitution
Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 angstrom resolution by electron cryomicroscopy and angular reconstitution
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DOI：
10.1006/jmbi.1997.1182
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发表时间：
1997-08-22
影响因子：
5.6
通讯作者：
vanHeel, M
中科院分区：
文献类型：
--
作者：
Orlova, EV;Dube, P;vanHeel, M
在液态氦气和单个颗粒图像处理下，使用电子冷冻显微镜和单个颗粒图像处理获得了钥匙孔lim lim蛋白1型血氰蛋白蛋白（KLH1）的三维重建。使用高对比度嵌入培养基，1％（w/v）葡萄糖和2％（w/v）钼酸铵（pH 7.0），使高分辨率电子显微照片能够接近焦点，即具有出色的传递高分辨率信息的同时，保持了足够的图像对比，以将图像中的单个大分子定位。当低通滤波到类似于45 Angstrom分辨率时，新的15 Angstrom分辨率重建与早期嵌入玻璃体冰的标本的胃血胶质蛋白的早期重建非常相似。该地图显示了很多细节，并揭示了这个非常大的圆柱体软体动物血胶蛋白中的许多新对称元素。完整的KLH1 DIDECAMER具有D5点组对称性，但在KLH1 deTameceric Half-Molecules内局部2倍轴已经出现了使KLH1 Decamer的壁的出现，尽管它仅具有精确的C5对称性dec骨头足动物血红蛋白。实际上，发现该六层腹腔底血液蛋白的每个层的外部都具有近似的D5对称性。局部2倍轴也将壁的“二聚体”“形态单元”和8 MDA KLH1分子的锁骨区域相关联。某些与局部对称性相关的表面基序最多可能存在于这种高度对称的圆柱形血胶蛋白的外壁60次。钥匙孔lig骨血胶蛋白在临床上用作免疫刺激剂。该血胶蛋白引起的非常强的免疫反应可能与局部对称成分的复杂层次结构有关。 （c）1997 Academic Press Limited。
A three-dimensional reconstruction of keyhole limpet hemocyanin type 1 (KLH1) has been obtained using electron cryomicroscopy at liquid helium temperatures and single particle image processing. The use of a high-contrast embedding medium, 1% (w/v) glucose and 2% (w/v) ammonium molybdate (pH 7.0), enables high-resolution electron micrographs to be recorded close to focus, i.e. with excellent transfer of high-resolution information, while maintaining enough image contrast to localise the individual macromolecules in the images. When low-pass filtered to similar to 45 Angstrom resolution, the new 15 Angstrom resolution reconstruction is very similar to the earlier reconstructions of gastropodan hemocyanins of specimens embedded in vitreous ice. The map shows much detail and reveals many new symmetry elements in this very large cylindrical molluscan hemocyanin. The full KLH1 didecamer has D5 pointgroup symmetry, yet within the KLH1 decameric half-molecules local 2-fold axes have emerged that make the wall of the KLH1 decamer, in spite of its having an exact C5 symmetry only, resemble the D5-symmetric wall of the decameric cephalopod hemocyanins. In fact, the outside of each tier of this six-tiered gastropodan hemocyanin was found to have an approximate D5 symmetry. Local 2-fold axes also relate the ''functional units'' within the dimeric ''morphological units'' of the wall and the collar areas of the 8 MDa KLH1 molecule. Certain local-symmetry-related surface motifs may be present up to 60 times on the outside wall of this highly symmetric cylindrical hemocyanin. Keyhole Limpet hemocyanin is used clinically as an immunostimulant. The very strong immune reaction elicited by this hemocyanin may be associated with its intricate hierarchy of local-symmetry components. (C) 1997 Academic Press Limited.