分子置换法在去B链羧端五肽胰岛素晶体结构测定中的应用

收集了一套去B链羧端五肽胰岛素(DPI)单斜晶体的X射线衍射数据。利用已知的胰岛素结构和DPI强度数据,应用分子置换法对测定DPI的晶体结构作了尝试。为了测定分子在晶胞中的取向和位置,在TQ-16和013计算机上编制了一套ALGOL程序。在算得的旋转函数图上没有找到突出的峰,用去掉B链前三个氨基酸残基和大部分表面侧链的胰岛素分子的原子坐标数据作试验,得到了改进的10～4图。在此基础上,采用不同的方法探索分子在单斜晶胞里的位置,最后得到了一个可能的分子在晶胞里的堆积模型。结果表明,胰岛素和DPI分子结构之间的差异可能较大,DPI晶体中晶体学二重轴联系的两个分子不能形成一个象三方二锌猪胰岛素晶体那样的二聚体。     

天花粉蛋白的第三种晶型-P2_1晶型

用平衡透析法得到了新的一种P2_1晶型的天花粉蛋白的单晶,其衍射能力优于2.5(?).晶体属单斜晶系,P2_1空间群,晶胞参数,a=73.4(?),b=74.7(?),c=87.9(?),β=97.7°;一个不对称单位含四个天花粉蛋白分子.文中还讨论了不同盐离子对天花粉蛋白分子在晶胞中堆积方式的影响.     

分子置换法在去B链羧端五肽胰岛素晶体结构测定中的应用

收集了一套去B 链羧端五肽胰岛素(DPI)单斜晶体的X 射线衍射数据。利用已知的胰岛素结构和DPI 强度数据,应用分子置换法对测定DPI 的晶体结构作了尝试。为了测定分子在晶胞中的取向和位置,在TQ-16和013计算机上编制了一套ALGOL 程序。在算得的旋转函数图上没有找到突出的峰,用去掉B 链前三个氨基酸残基和大部分表面侧链的胰岛素分子的原子坐标数据作试验,得到了改进的10～4(?)图。在此基础上,采用不同的方法探索分子在单斜晶胞里的位置,最后得到了一个可能的分子在品胞里的堆积模型。结果表明,胰岛素和DPI分子结构之间的差异可能较大,DPI 晶体中晶体学二重轴联系的两个分子不能形成一个象三方二锌猪胰岛素晶体那样的二聚体。     

富硒螺旋藻中含硒藻蓝蛋白的纯化、结晶及初步晶体学研究

从富硒螺旋藻中提取含硒藻蓝蛋白,经凝胶色谱和离子交换色谱纯化,应用悬滴气相扩散法,采用(NH4)2SO4和PEG4000作沉淀剂,获得了该蛋白质晶体的两种晶型.晶型Ⅰ属于单斜晶系,晶胞参数a=10.80 nm,b=11.70 nm,c=18.40 nm,β=90.2°,晶体空间群属于P21.单位晶胞中每个晶体学不对称单位含12个(αβ)单体,晶体衍射的最高分辨率达0.28 nm.晶型Ⅱ为六方晶系,晶胞参数为a=b=15.5 nm,c=4.03 nm,晶体空间群属于P63.晶体衍射的最高分辨率达0.28 nm.单位晶胞中每个晶体学不对称单位含1个(αβ)单体.对分子在晶体中的可能堆积方式进行了讨论.     

江浙蝮蛇毒碱性磷脂酶A2正交晶型Ⅰ--0.25nm分辨率晶体结构

江浙蝮蛇毒碱性磷脂酶A2具有强烈的溶血及抗凝血活性.运用刚体修正技术获得了正交晶型Ⅰ中分子的精确旋转与平移参数.采用非晶体学二重对称性制约的最小二乘修正方法在0.6～0.25 nm分辨率范围内进行了结构精化.最终的晶体学R因子为20.1%, 键长、键角与标准值的均方根偏差分别为0.0013 nm和1.55°.与正交晶型Ⅱ结构比较表明,二者除了β-折叠部位与Ca2+结合部位构象存在小的差别外,磷脂酶A2分子主体构象极其相似.2种晶型由不对称单位中2个分子形成的二体也是类似的.但是,二体中1个单体分子的相对取向有5.5°的差别,提示二体结合面有一定柔性.晶型Ⅰ二体较晶型Ⅱ二体在晶胞中的堆积更紧密.     

B链N端去二肽(B1～2)C端去五肽(B26～30)胰岛素晶体结构的分子置换法研究

以B链羧端去五肽胰岛素(DPI)结构为模型,应用分子置换法对B链N端去二肽(B1～2)C端去五肽(B26～30)胰岛素(DesB1～2DPI)晶体结构进行了研究.DesB1～2DPI晶体单位晶胞中每个结晶学不对称单位包含1个DesB1～2DPI分子.交叉旋转函数和平移函数搜索均找到了明显突出的峰,确定了DesB1～2DPI分子在晶胞中的取向和位置.进一步的三维模型重建和结构精化结果巩固了DesB1～2DPI的分子置换法研究的正确性.     

B链羧端去七肽(B24～B30)胰岛素:一种新晶型的结构

B链羧端去七肽 (B2 4～B30 )胰岛素 (DHPI)在同样的晶体生长条件下可以得到两种晶型 ,即晶型A和晶型B .测定了 0 .2nm分辨率B型DHPI(DHPI B)的晶体结构 .B型DHPI分子的整体结构与已报道的A型DHPI分子 (DHPI A)很相似 ,但局部区域的构象以及分子在晶胞中的堆积存在较大的差异 .DHPI的晶体结构显示了 ,在结晶条件下 ,一个独立区内 2个DHPI单体分子构成了一个DHPI二体 .DHPI A和DHPI B因二体形成而包埋的作用面 (作用面Ⅱ )面积分别达到 1 8.2 0和 1 6 .95nm2 ,这一面积在目前所有胰岛素及其类似物的晶体结构中是最大的 .仔细考察胰岛素及其截断体类似物的晶体结构可以发现 ,该作用面广泛存在于这些分子的缔合作用中 .研究结果表明 ,作用面Ⅱ在同一晶体生长条件下 2种晶型的形成中起关键作用 .     

沉淀剂类型对蛋白质晶体分子堆积的影响

以不对称单位只有一个分子的牛胰核糖核酸酶和T4溶菌酶晶体为材料,着重研究了无机盐、有机溶剂和PEG三类不同的沉淀剂对晶体分子堆积的影响,经研究发现两种蛋白质中用无机盐做沉淀剂的晶型几乎都含有面积较大的二次轴对称接触面和较少的相邻分子数,同时其含有的参与接触的非极性残基集中分布于二次轴对称接触面,而盐键则在二次轴对称接触面上分布稀少。用有机溶剂作沉淀剂的晶型却含有面积较小的非二次轴对称接触面和较多的相含分子数,而参与接触的非极性残基和直键在各个非二次轴对称接触面上随机分布,用PEG作沉淀剂的晶型其分子堆积特征总体上类似于用有机溶剂作沉淀剂的晶型,但个别晶型具有与用无机盐做沉淀剂的晶型相似的分子堆积特征,以上结果提示,用三类沉淀剂得到的不同的分子堆积特征可能与三类沉淀剂不同的诱导结晶机理密切相关。     

晶态下氯化氨甲酰胆碱结构研究

应用X衍射分析确定了晶态下氯化氨甲酰胆碱分子的结构,它们形成以氢键联结的沿晶胞c方向延伸的两个互为对映、相间分布的螺旋型多聚体结构。     

江浙蝮蛇毒碱性磷脂酶A2正交晶型Ⅰ——0.25nm分辨率晶体结构

江浙蝮蛇毒碱性磷脂酶A₂ 具有强烈的溶血及抗凝血活性 .运用刚体修正技术获得了正交晶型Ⅰ中分子的精确旋转与平移参数 .采用非晶体学二重对称性制约的最小二乘修正方法在 0 .6～ 0 .2 5nm分辨率范围内进行了结构精化 .最终的晶体学R因子为 2 0 .1 % ,键长、键角与标准值的均方根偏差分别为 0 .0 0 1 3nm和 1 .5 5° .与正交晶型Ⅱ结构比较表明 ,二者除了β -折叠部位与Ca ²⁺结合部位构象存在小的差别外 ,磷脂酶A₂分子主体构象极其相似 .2种晶型由不对称单位中 2个分子形成的二体也是类似的 .但是 ,二体中 1个单体分子的相对取向有 5 .5°的差别 ,提示二体结合面有一定柔性 .晶型Ⅰ二体较晶型Ⅱ二体在晶胞中的堆积更紧密 .     

Molecular dynamics simulations of a double unit cell in a protein crystal: volume relaxation at constant pressure and correlation of motions between the two unit cells

Eight molecular dynamics simulations of a double crystal unit cell of ubiquitin were performed to investigate the effects of simulating at constant pressure and of simulating two unit cells compared to a single unit cell. To examine the influence of different simulation conditions, the constant-pressure and constant-volume simulations were each performed with and without counterions and using two different treatments of the long-range electrostatic interactions (lattice-sum and reaction-field methods). The constant-pressure simulations were analyzed in terms of unit cell deformation and accompanying protein deformations. Energetic and structural properties of the proteins in the simulations of the double unit cell were compared to the results of previously reported one-unit-cell simulations. Correlation between the two unit cells was also investigated based on relative translational and rotational movements of the proteins and on dipole fluctuations. The box in the constant-pressure simulations is found to deform slowly to reach convergence only after 5-10 ns. This deformation does not result from a distortion in the structure of the proteins but rather from changes in protein packing within the unit cell. The results of the double-unit-cell simulations are closely similar to the results of the single-unit-cell simulations, and little motional correlation is found between the two unit cells.     

Conformation and packing properties of phosphatidic acid: The crystal structure of monosodium dimyristoylphosphatidate

The conformation and molecular packing of monosodium 1,2-dimyristoyl-sn-glycerophosphate (DMPA) has been determined by single crystal analysis (R = 0.107). The lipid crystallizes in the space group P2₁ with unit cell dimensions: $\text{a = 5.44, b = 7.95, c = 43.98}\text{A}\text{?}$ and β = 114.2°. The two molecules of the unit cell are related by a two-fold screw axis and pack tail-to-tail in a bilayer structure. The monosodium phosphate group packs with rather a small cross-section (24 Ų) relative to the two hydrocarbon chains. This unbalance in packing cross-section is overcome by an interdigitation of the phosphate head groups of adjacent bilayers and the formation of a single, common phosphate group layer at the bilayer interfaces. The phosphate groups are linked by hydrogen bonds to linear strands which laterally are separated by strands of sodium ions. The conformation of the molecules differs from that of other phospholipids. The glycerol chain is oriented parallel (instead of perpendicular) to the layer surface and the parallel stacking of the hydrocarbon chains is achieved by a bend of the γ-chain (instead of the β-chain). Otherwise the conformation of the glycerol dicarboxyl ester group displays the same preferred features as generally found in glycerophospholipids. The hydrocarbon chains pack according to the triclinic (T_∥) packing mode. The interaction and packing principles of the phosphate head group are discussed in relation to the structural behaviour of phosphatidic acid.     

Interactions and space requirement of the phosphate head group of membrane lipids: The single crystal structures of a triclinic and a monoclinic form of hexadecyl-2-deoxyglycerophosphoric acid monohydrate

The crystal structures of a triclinic form (HPA1) and a monoclinic form (HPA2) of hexadecyl-2-deoxyglycerophosphoric acid monohydrate were determined by single crystal analysis. The unit cell dimensions for HPA1 are $\text{a = 4.75, b = 5.72, c = 44.36}\text{A}\text{?}$ and α = 91.0, β = 101.5, γ = 100.5° (P$\text{1}$) and for HPA2, $\text{a = 4.75, b = 5.72, c = 88.72}\text{A}\text{?}$ and γ = 100.8° (P2₁). In both structures the molecules are fully extended and pack tail-to-tail in bilayers with tilting (47°) hydrocarbon chains. In HPA2, however, the chain tilt alternatingly changes direction in adjacent bilayers, giving rise to a doubled unit cell which spans two bilayers. The dihydrogen phosphate groups interact by hydrogen bonds and are arranged in rows. Laterally between these phosphate rows the water molecules are accommodated producing a compact two-dimensional network of hydrogen bonds. The packing cross-section in the layer plane of the dihydrogen phosphate monohydrate group is 26.7 Ų in both structures. The hydrocarbon chains pack according to the triclinic (T|) chain packing mode. In HPA2, however, the chain packing is somewhat less compact with accounts for a 2% increase in the molecular volume. In both structures the ether oxygen is accommodated into the hydrocarbon matrix without distortion of the chain packing.     

MOLPACK: molecular graphics,for studying the packing of protein molecules in the crystallographic unit cell

A graphics program, MOLPACK, has been developed on the Silicon Graphics IRIS-4D computer system for displaying the packing of proteins in the crystallographic unit cell. In addition to the normal viewing operations of rotation, translation and scaling, the program has the ability to translate molecules along the cell axes while maintaining their crystallographic equivalent positions within the unit cell. This allows the user to observe the packing of protein molecules generated by molecular replacement, to create a new packing model or to locate an unknown molecule. A special feature of the program is that up to four independent molecules can be manipulated in the asymmetric unit.     

The crystal structure of Bombyx mori silk fibroin at the air–water interface

A new crystalline polymorph of Bombyx mori silk, which forms at the air–water interface, has been characterized. A previous study found this structure to be trigonal, and to be distinctly different than the two previously observed silk crystal structures, silk I and silk II. This new structure was named silk III. Identification of this new silk polymorph was based on evidence from transmission electron microscopy and electron diffraction, coupled with molecular modeling. In the current paper, additional data enables us to refine our model of the silk III structure. Some single crystal electron diffraction patterns indicate a deviation in symmetry away from a perfect trigonal unit cell to monoclinic unit cell. The detailed shape of the powder diffraction peaks also supports a monoclinic cell. The monoclinic crystal structure has an nonprimitive unit cell incorporating a slightly distorted hexagonal packing of silk molecular helices. The chains each assume a threefold helical conformation, resulting in a crystal structure similar to that observed for polyglycine II, but with some additional sheet-like packing features common to the threefold helical crystalline forms of many glycine-rich polypeptides. © 1997 John Wiley & Sons, Inc. Biopoly 42: 705–717, 1997     

Calculations on crystal packing of a flexible molecule, Leu-enkephalin

Crystal packing calculations have been carried out on a substantial number of conformations of Leu-enkephalin; namely, those obtained both from crystal structures and from energy minimizations on isolated molecules, and with and without waters of crystallization. The known crystal structures represent the most energetically stable packings found. The conformations of the enkephalin molecules in the crystal are not the most stable for an isolated molecule; i.e. intermolecular interactions force the isolated molecule to change conformation in order to achieve a small packing volume and an optimal packing energy in the crystal. It is found that the packing energy of an enkephalin molecule is a reasonably smooth function of its molecular volume in the unit cell, if structures with intermolecular hydrogen bonding are excluded, and is substantially independent of other details of the molecular conformation or of the crystal packing. Hydrogen bonding provides additional stabilization of the crystal structure, and would likely permit crystallization of the system if it is sufficiently dense. Solvent molecules further stabilize the structure when they can also provide intermolecular hydrogen bonds.     

Crystal structures of cyclomaltohexaose (alpha-cyclodextrin) complexes with p-bromophenol and m-bromophenol.

Crystal structures of cyclomaltohexose (alpha-cyclodextrin) complexes with p-bromophenol and m-bromophenol have been determined by single-crystal X-ray diffraction. The space group of the alpha-cyclodextrin-p-bromophenol complex is P2(1)2(1)2(1) with unit cell dimensions of a = 15.318(3), b = 24.733(3), c = 13.457(2) A, and that of the alpha-cyclodextrin-m-bromophenol complex is P2(1)2(1)2 with unit cell dimensions of a = 25.858(7), b = 27.263(8), c = 8.145(3) A. In crystals, the alpha-cyclodextrin-p-bromophenol complex and the alpha-cyclodextrin-m-bromophenol complex form a layer-type and a channel-type molecular packing structure, respectively. The intermolecular hydrogen-bond interactions of the hydroxyl groups of bromophenols are closely related to the molecular packing structure.     

X-ray study of chymosin. I. Molecular replacement at a 3 angstroms resolution

The determination of the three dimensional structure of chymosin at 3 A resolution by molecular replacement method is described. The rotation functions for various aspartic proteases were calculated and combined results were used for the refinement of orientational parameters of chymosin molecules in the unit cell. The interpretation of Crowther-Blow translation function map with packing consideration enable to place correctly the molecules in the chymosin unit cell. Several difference Fourier syntheses for chymosin were calculated and differences between pepsin and chymosin structures were detected.     

Crystallization and subunit structure of canine myeloperoxidase

Three crystal forms of canine myeloperoxidase are described. An orthorhombic form in space group P2(1)2(1)2(1) has unit cell dimensions: a = 108.3 A (1 A = 0.1 nm) b = 205.9 A and c = 139.9 A. A trigonal form in space group P3(1)21 or P3(2)21 has unit cell dimensions: a = b = 138.9 A and c = 145.2 A. A monoclinic form in space group C2 has unit cell dimensions: a = 117.2 A, b = 96.9 A, c = 131.4 A and beta = 116.3 degrees. Unusual features in the diffraction patterns of the monoclinic form place restrictions on the molecular packing in the crystal. The proposed model for the molecular packing requires that the myeloperoxidase molecule consist of two identical or near-identical halves. In the intact molecule these halves may be related either by a crystallographic dyad axis or by an approximate dyad axis in which one subunit is translated relative to the other by 3.2 A along the symmetry axis. The trigonal crystal form appears most suitable for high-resolution X-ray structural analysis.     

Molecular replacement study on form-B monoclinic crystal of insulin

The form-B monodinic insulin crystal was obtained from the sodium citrate buffer with 1% zinc chloride, keeping phenolic content between 0.76% and 1.25%. Its space group is P21, cell constants are: a = 4.924nm, b=6.094nm, c=4.818nm, β=95.8°. There are 6 insulin molecules which form a hexamer. The initial phase was obtained by using rotation function program of X-PLOR program package and molecular packing program of our laboratory. The molecular model was chosen from 4 zinc bovine insulin hexamer. After the preliminary refinement by using the rnacromolecular rigid body refinement technique, the molecular model was further refined and adjusted by using the energy-minimizing stereochemically restrained least-squared refinement on the difference Fourier maps. The finial R-factor is 214% at 0.3nm resolution, the r.m.s. deviations from standard bond length and bond angle are 0.0022nm and 4.7°, respectively.