Koepke Juergen, Scharff Eileen I, Lücke Christian, Rüterjans Heinz, Fritzsch Günter
Max-Planck-Institute of Biophysics, Department of Molecular Membrane Biology, Marie-Curie-Strasse 15, D-60439 Frankfurt/Main, Germany.
Acta Crystallogr D Biol Crystallogr. 2003 Oct;59(Pt 10):1744-54. doi: 10.1107/s0907444903016135. Epub 2003 Sep 19.
The X-ray crystal structure of squid-type diisopropylfluorophosphatase (DFPase) has been refined to a resolution of 0.85 A and a crystallographic R value of 9.4%. Crystal annealing improved both the mosaicity and resolution of the crystals considerably. The overall structure of this protein represents a six-bladed beta-propeller with two calcium ions bound in a central water-filled tunnel. 496 water, two glycerol and two MES buffer molecules and 18 PEG fragments of different lengths could be refined in the solvent region. 45 of the 314 residues have been refined with alternative orientations. H atoms have been omitted from disordered residues. For the residues of the inner beta-strands, H atoms are visible in a normal F(o) - F(c) difference map of a hydrogen-deficient structure model. The 208 most reliable residues, without disorder or reduced occupancy in their side chains, were finally refined without restraints. A subsequent full-matrix refinement cycle for the positional parameters yielded estimated standard deviations (e.s.d.s) by matrix inversion. The thus calculated bond lengths and bond angles and their e.s.d.s were used to obtain averaged bond lengths and bond angles, which were compared with the restraints applied in the preceding refinement cycles. The lengths and angles of the hydrogen bonds inside the antiparallel beta-sheets of the DFPase structure were compared with data averaged over 11 high-resolution protein structures. Torsion angles were averaged according to angle types used as restraints in X-PLOR and CNS and subsequently compared with values obtained from 46 high-resolution structures. Side-chain torsion angles were also classified into rotamer types according to the Penultimate Rotamer Library. Moreover, precise dimensions for both Ca(2+)-coordination polyhedra could be obtained and the coordination of one Ca(2+) ion by an imidazole N atom was confirmed. This statistical analysis thus provides a first step towards a set of restraints that are founded completely on macromolecular data; however, 10-20 additional protein data sets of comparable accuracy and size will be required to obtain a larger statistical base, especially for side-chain analysis.
鱿鱼型二异丙基氟磷酸酯酶（DFPase）的X射线晶体结构已精修至分辨率为0.85埃，晶体学R值为9.4%。晶体退火显著改善了晶体的镶嵌性和分辨率。该蛋白质的整体结构呈现为一个六叶β-螺旋桨，两个钙离子结合在中央充满水的通道中。在溶剂区域可精修496个水分子、两个甘油分子和两个MES缓冲分子以及18个不同长度的PEG片段。314个残基中的45个已用替代取向进行精修。无序残基中的氢原子已被省略。对于内部β-链的残基，在缺氢结构模型的正常F(o)-F(c)差值图中可见氢原子。最终对208个最可靠的残基进行了无约束精修，这些残基的侧链无无序或占有率降低的情况。随后对位置参数进行的全矩阵精修循环通过矩阵求逆产生了估计标准偏差（e.s.d.s）。由此计算出的键长和键角及其e.s.d.s用于获得平均键长和键角，并与之前精修循环中应用的约束条件进行比较。将DFPase结构反平行β-折叠内部氢键的长度和角度与11个高分辨率蛋白质结构的平均数据进行了比较。扭转角根据在X-PLOR和CNS中用作约束条件的角度类型进行平均，随后与从46个高分辨率结构获得的值进行比较。侧链扭转角也根据倒数第二个旋转异构体库分类为旋转异构体类型。此外，可获得两个Ca(2+)配位多面体的精确尺寸，并证实了一个Ca(2+)离子与一个咪唑N原子的配位。因此，这种统计分析为完全基于大分子数据的一组约束条件迈出了第一步；然而，需要另外10 - 20个具有可比准确性和规模的蛋白质数据集来获得更大的统计基础，特别是用于侧链分析。
