3-D CRYSTAL STRUCTURE:VERTEBRATE MITOCHONDRIA COMPLEX II

X射线衍射数据是蛋白质原子分辨率三维结构信息的主要来源。 虽然大量的信息是潜在的，从这项技术，它的应用需要的能力，以准备晶体的大小和顺序适合X射线分析。 这对于膜蛋白来说尤其困难。络合物II（琥珀酸酯：泛醇氧化还原酶）是一种膜蛋白复合物，其将电子从琥珀酸汇集到线粒体呼吸链中。 呼吸链负责生物氧化和以跨线粒体内膜的质子电化学电位梯度的形式释放的能量的保存。 然后，来自该梯度的能量用于合成ATP或通过跨膜运输物质来做功。 许多线粒体肌病和中枢神经系统疾病已被证明是由于线粒体电子传递链的缺陷，在某些情况下，在复合物II。在我们研究线粒体bc1复合体的过程中，我们从许多脊椎动物来源中制备并溶解了线粒体。 我们已经学会了制备复合物II作为细胞色素bc1制备的副产品。 在鸡酶的情况下，我们已经能够使酶结晶。 晶体可以快速冷冻，用于低温数据收集，衍射高达3.5埃单位。 这些数据可以在正交晶格上索引，晶胞边缘为69.036 x 83.735 x 290.214埃单位，系统缺失表明空间群为P212121。 似乎可以合理地预期，随着尺寸和镶嵌性的改善，晶体将超过3.0埃单位，这将足以建立蛋白质的原子模型，一旦数据被分阶段。建议（a）通过使用重结晶和引晶技术以及更仔细地控制结晶条件，提高晶体在尺寸和镶嵌性方面的质量。 (B)收集衍射数据并对其进行定相，或者通过使用来自E. coli或W. succinogenes，或（在这些结构都不可用的情况下）通过同晶衍生物和多波长反常色散技术，使用重原子衍生物和络合物的固有铁。 (C)在电子密度图中建立蛋白质的原子模型，并根据衍射数据对其进行改进，以提交给蛋白质数据库。

X-ray diffraction data is the main source of 3-dimensional structure information at atomic resolution for proteins. While a great deal of information is potentially available from this technique, its application requires the ability to prepare crystals of size and order suitable for X-ray analysis. This has proved especially difficult for membrane proteins. Complex II (succinate: ubiquinol oxidoreductase) is a membrane protein complex, which funnels electrons from succinate into the mitochondrial respiratory chain. The respiratory chain is responsible for biological oxidation and for conservation of the energy released in the form of a proton electrochemical potential gradient across the mitochondrial inner membrane. Energy from this gradient is then used to synthesize ATP or to do work by transporting substances across the membrane. A number of mitochondrial myopathies and CNS disorders have been shown to be due to defects in the mitochondrial electron transport chain and in some cases in complex II. In the course of our work on the mitochondrial bc1 complex we have prepared and solubilized mitochondria from a number of vertebrate sources. We have learned to prepare complex II as a by-product of the cytochrome bc1 preparation. In the case of the chicken enzyme, we have been able to crystallize the enzyme. The crystals can be flash-frozen for cryogenic data collection with diffraction up to 3.5 Angstrom units. The data can be indexed on an orthorhombic lattice with unit cell edges 69.036 x 83.735 x 290.214 Angstrom units, and systematic absences suggest the spacegroup P212121. It seems reasonable to expect that with improvement in size and mosaicity the crystals will diffract beyond 3.0 Angstrom units, which would be sufficient to build an atomic model of the protein once the data is phased. It is proposed to (a) Improve the quality of the crystals with respect to size and mosaicity, by using recrystallization and seeding techniques and more carefully controlling the crystallization conditions. (B) Collect diffraction data and phase it, either by molecular replacement using the coordinates from fumarate reductase of E. coli or W. succinogenes, or (in case neither of those structures is available) by isomorphous derivative and multiwavelength anomalous dispersion techniques using heavy atom derivatives and the intrinsic irons of the complex. (C) Build an atomic model of the protein into the electron density map and refine it against the diffraction data, for submission to the protein data bank.