X-RAY STUDIES ON THE STRUCTURE OF DES-PENTAPEPTIDE INSULIN AT 2.4  RESOLUTION——SOLUTION OF THE PHASE PROBLEM USING THE ANOMALOUS SCATTERING OF CADMIUM AND THE DERIVATION OF A MODEL STRUCTURE

The three dimensional structure of des-pentaptide insulin has been solved at a resolu-tion of 2.4A. The structure determination was achieved without the use of heavy atom de-rivatives, by using only the phase information obtained from the seattering (both real andimaginary) of X-rays by cadmium ions present in the native crystal. Using this phase in-formation, a map, with figure of merit 0.56, was produced which enabled an initial modelof the protein to be constructed. Because of the novelty of this approach to the solutionof the phase problem the procedures used are explained in some detail.     

2.4分辨率去五肽胰岛素结构的X射线研究——DPI分子及其同胰岛素的关系

本文描述了单体胰岛素类似物去五肽(B26—B30)胰岛素的结构。同三方二锌胰岛素结构的详细比较说明,DPI虽然在结构上有一些大的改变,但是基本的二级结构单位仍然完整地保持着。像胰岛素的其他晶形那样,DPI结构比较接近于二锌胰岛素结构中的分子Ⅰ。除了结构表面的改变之外,在分子中心还有一些微妙和广泛的改变。分子在晶体中紧密地堆积,有着许多不同的接触,包括蛋白质-镉相互作用的复合网及大量以水为媒介的接触。分子表面大批疏水基团绕蛋白质群集而成一厚带。晶体结构具有良好的有序性,某些侧链的清晰度和水分子的确定性优于在更具活动性的三方二锌胰岛素六体晶体所得的结果。     

2.4分辨率去五肽胰岛素结构的X射线研究——结构的修正

本文报道起始模型对于X射线实验观测量和已知立体化学观测量作约束参数最小二乘修正的结果.经修正和扩展的模型,不仅包括DPI分子的全部非氢原子和一个镉原子,而且还加进49个水分子.由反常散射和模型信息概率相联合得到相角,各项系数按相角可靠性加权,其2F_o-F_c Fourier综合给出清晰的结果,无明显的对试用模型的偏向.最后得到的模型有合理的立体化学,同理想键长的均方根偏离是0.027A;同观测X射线散射相当吻合,对 10—2.4A分辨率的全部数据其偏离因子R=0.182.     

1.5分辨率去五肽(B26—30)胰岛素晶体中的水结构

本文报道1.5分辨率去五肽(B 26—30)胰岛素晶体中的水结构。根据扩充F_0的最后的(2F_o—F_c)Fourier图的检验,所讨论的水模型包括81个水分子(电子密度>0.4 e/~3),约占晶体溶剂的三分之二。以2.4—3.2为氢键范围,51个水分子同蛋白质原子成氢键,占水的63％,其中有12个以单水桥接相邻蛋白质分子,更多的以双水桥接相邻蛋白质分子。在蛋白质分子间的一长狭缝中有一个紧凑的水网,两头两个Cd原子通过三个水分子(配位体)像木桩一样支撑着这个水网,表明Cd原子和水网在分子密堆积中的重要作用。     

2.4埃分辨率去五肽胰岛素结构的X射线研究——用镉反常散射解相角问题和模型结构的导出

本文证明2.4埃分辨率去五肽胰岛素三维结构已经解得.这个结构的测定没有使用重原子衍生物,而只用了由本晶体中镉离子的X射线散射(实部和虚部)获取的相角信息.这一相角信息具有0.56的总品质因数,由它计算的电子密度图可用来建立起蛋白质的初始模型.由于解相角问题所用方法目前仍属于一种新的方法,我们在文中将予以适当阐述。     

去B链羧端六肽(B25—30)胰岛素初步结晶学研究

去B链羧端六肽(B25—30)胰岛索,采用悬挂液滴气相扩散法,在柠檬酸纳缓冲液pH 5.8;含有丙酮和氯化钠体系中,获得可供X射线晶体结构分析用的单晶体,其衍射分辨率在1.5以上,晶体外形为三角形。属于单斜晶系,空间群为C2,晶胞参数为α=42.60,b=37.87,c=27.118,β=125.35°。其结构分析工作正在进行中。     

X-RAY STUDIES ON THE STRUCTURE OF DES-PENTAPEPTIDE INSULIN AT 2.4 RESOLUTION——THE REFINEMENT OF THE STRUCTURE

The determination of an initial model for the crystal structure of des-pentapeptide in-sulin, using only the phase information obtained from the scattering of cadmium ions inthe native crystal, has been described. This paper describes the refinement of this modelagainst both the experimental X-ray observations and the a priori stereochemical observationsusing the method of restrained parameter least squares. The corrected and extended modelincluded not only all the non-hydrogen atoms of the protein and one cadmium ion, but alsosome 49 water molecules. Fourier syntheses using amplitudes of 2F_o-F_c, phase angles prob-abilistically combined from the anomalous scattering and model phase information, witheach term weighted according to its reliability gave clear results without detectable bias to-wards the current model. The final model has acceptable stereochemistry (root mean squaredeviation from ideal bond lengths 0.027 A), and predicts the observed X-ray scattering sat-isfactorily (R = 0.182 for all dat     

PRELIMINARY CRYSTALLOGRAPHIC ANALYSIS OF DESHEXAPEPTIDE (B25—B30) INSULIN

Satisfactory single crystals of deshexapeptide(B25—B30) insulin for X-ray crystal structure analysis have been grown in citrate buffer by the method of hanging-drop gas phase diffusion. The crystal belongs to the monoclinic system with space group C2. The unit cell constants are α=42.6, b=37.9, c=27.2, β=125.4 and there is only one molecule of deshexapeptide insulin in an asymmetric unit.     

WATER STRUCTURE IN DESPENTAPEPTIDE (B26—B30) INSULIN CRYSTAL AT 1.5  RESOLUTION

The water structure in despentapeptide (B26-B30) insulin crystal is presented in this paper. Ac-cording to inspection of the final F_0-extended (2F_0-F_c) Fourier map, 81 water molecules (about twothirds of solvent) with electron density greater tLan 0.4 e/A~3 are included in the water model to bediscussed. In a hydrogen bond length range of 2.4--3.2 A, 51 water molecules, that is, 63% of thetotal 81, are hydrogen bonded to protein, of which 12 link the adjacent protein molecules through one-water bridges and more through two-water bridges. There is a tight water network in a long crevice be-tween protein molecules; two Cd atoms, like two piles, prop up the network through three water molecules(Cd ligands) at either end of the network, indicating that Cd ions and water networks play an importantrole in close packing of protein molecules in the crystal.     

REFINEMENT OF THE STRUCTURE OF DESPENTAPEPTIDE (B26—30) INSULIN AT 1.5 RESOLUTION

The refinement of the structure of despentapeptide (B26-20) insulin (DPI) at 1.5 Aresolution is described in the present paper. Against the X-ray diffraction data of newlygrown crystals, the restrained parameter least-squares refinement of the structure alter-nated with model-rebuilding. The original 2.4A resolution model has been thoroughlyrefined and parts of it have been eonsiderably revised. The cadmium coordination struc-ture has been well determined, and appears to be a nearly ideal octahedron. After refine-ment 84 water oxygen atoms have been included in the final model; this gives us detailedinformation for studies on the water structure in the DPI crystal. The final crystal-lographic reliability index R value was 0.144 for 5678 unique observable reflexions (F_o great-er than 1.5σ (F_o)) in the 10-1.5A range; the refined model has a root-mean-squaredeviation from standard bond lengths of 0.04A.