用原子力显微镜（AFM）进行蛋白质晶体生长及成核研究
摘要
生物大分子(包括蛋白质、核酸和病毒等)对生物体的生命活动起着十分重要的作用，这是由它们具有特殊的空间结构所致。随着生物学领域对精确的生物大分子结构需求的日益增加，尤其是利用单晶X射线衍射法得到的三维结构，使得研究生物大分子晶体的生长越来越受到重视。然而，生物大分子的结晶相当困难，从某种意义上讲这是生物大分子结构测定的限速因素。目前对生物大分子的晶体生长机理、成核的过程、基本的热力学和动力学参数等的认识很少，所以在晶体的缺陷、分子在晶格中的定向力和分子间键连作用以及溶液中传质过程方面的认识也很有限。因此，进行的生物大分子晶体生长的研究就显得非常必要。目前已有一些技术应用于研究生物大分子晶体的生长机理中，如光散射技术(QELS)和原子力显微镜(AFM)等，而迄今为止，AFM仍为进行这一研究的非常重要的工具。
本论文通过AFM实验主要进行了部分蛋白质晶体的生长过程的研究，包括杜仲抗真菌蛋白(Eucommia antifungal protein, 简称EAFP)、天花粉蛋白(Trichosanthin, 简称TCS)、天麻抗真菌蛋白(Gastrodia elata antifungal protein, 简称GAFP)，并取得了一定进展，有一些新的发现：
1．EAFP在其水溶液中能形成一种环状聚集体，在40mg/ml的高浓度时其直径大小从100nm到300nm左右， 而在低浓度(5mg/m/l)时只有10-25nm左右，且数量较少。有的形成一个完整的环，有的环正在形成。这说明EAFP分子间本身有着较强的相互作用，能自发地在水溶液中进行聚集，且随溶液的浓度变小而聚集能力降低。
2．EAFP晶体的生长速度很快，通常只需要数小时就可长到较大尺寸(约1mm)，要比普通的蛋白质晶体生长快很多倍， 而且沿着晶体学轴向的生长速率也各不相同，表现出明显的各向异性；且生长的速率还与蛋白质溶液的过饱和度密切相关，过饱和度较高时(大于1.78)生长极为快速，难以进行速率的测量；当过饱和度为1.45时, EAFP晶体的{100}面上的生长速率b方向上平均为12 nm/s, c方向上平均为24.2 nm/s；当过饱和度为1.15时, 其生长速率b方向上为4.1nm/s；c方向上为8.8nm/s, 而此时，其生长速率仍与报道的其他蛋白质晶体生长速率基本相当，这些都说明EAFP晶体具有很快的生长速率。
3．实时原位AFM研究EAFP单斜晶体{100}面的生长过程结果表明EAFP晶
体生长并非以单一的生长模式进行，而是随生长条件的变化采取不同的生长特征，有以下因素可能与EAFP的晶体快速生长机制相关：① 多重不对称二维岛堆垛生长源；② 活泼的不对称螺旋错位生长源(主要以不对称的双左旋为主，以不断出现的多个无规的右旋为次)；③ 台阶按编组生长的动态特性。
4．EAFP在低过饱和度下的螺旋生长过程中会出现台阶的合并，从而表现出单-双的螺旋台阶， 在小范围扫描中捕获到了这种现象，使得对螺旋生长台阶的合并过程有了更加深入的认识；并且台阶的生长受到沿晶体学b轴方向上的21螺旋对称的控制；当台阶生长至第八到第九层时，螺旋台阶会自动地形成完整的生长层，这充分说明了EAFP分子或分子簇具有良好的自身调节能力，这样在晶体生长过程中，晶体的缺陷减少，晶体质量提高，这也可说明EAFP晶体的x射线衍射能力很强的原因。
5．通过对EAFP晶体中各种相互作用分析表明：一个EAFP分子与周围的相邻分子间有218个残基间的相互作用，其中有18个是强的相互作用，有72个较强作用(间距小于4埃), 这些作用大部分与晶体学c方向上的作用相关, 说明在c方向上作用较强。由于这种差别，使得EAFP在结晶过程中，当分子受到不同方向上的作用力时，便形成了表面形貌的不对称，进而影响到晶体的外貌。另外，通过大分子结构分析XPLOR(NIH版)对EAFP在溶液中和在晶格中的分子能量的较为详尽的分析表明，尽管两种状态下的EAFP分子骨架差别不大(主要是侧链Arg残基伸展方向不同)，但晶格中的EAFP分子采用较为有利的构象，使结晶过程能够顺利进行；且沿结晶学不同方向上分子间的相互作用能（尤其是氢键能的差别）也不同，沿c方向上要比沿b方向上强约1/3，这也与AFM结果分析相一致。
6．发现天花粉晶体所有表面上没有螺旋位错或台阶生长，只在其{001}面上发现明显的非对称线性的3D核进行生长。这些线性聚体均沿着晶体学<110>姆较蚪信帕校蚁嗷テ叫校饪伤得魈旎ǚ鄯肿蛹湓谡飧龇较蛏嫌凶沤锨康南嗷プ饔茫沟梅肿友刈耪飧龇较蛉菀捉芯奂欢杂贕AFP晶体，原位实时动态的原子力显微成像初步的研究发现在其晶体的{001}表面晶体生长较为缓慢，没有发现台阶和螺旋位错生长现象，而是以分子聚集体的形式，慢慢从溶液中沉积到晶体表面，初步确定为法向生长(normal growth)。
由于蛋白质分子本身和蛋白质晶体生长体系的复杂性，本文的工作仅针对所研究对象进行讨论，进一步更深入的研究尚在进行中。
Biomolecules(e.g., proteins, nucleic acids and viruses) with specific structures play very important roles to many biological processes. To meet increasing demands of precise biomolecular 3-D structures,especially protein structures by X-ray diffraction, it is more and more necessary to study their crystal growth. However,growing protein crystals is very difficult and less knowledge of biomolecular crystal growth mechanism, process of nucleic formation, basic thermodynamic and kinetic parameters aren’t known as well as crystal defects, molecular orientable forces, etc. In order to learn their growth mechanisms, some technologies have been used such as Quasi-Elastic Light Scaterring(QELS) and Atomic Force Microscopy (AFM), and so far AFM is one of the most important tools. In this dissertation some protein crystal growth have mainly been studied by AFM such as Eucommia antifungal protein(EAFP), Trichosanthin(TCS), Gastrodia elata antifungal protein(TCS),especially on EAFP crystals.
1．In the solution EAFP molecules can form ring-like clusters(about 40-300nm) at higher concentration(40mg/ml) and about 10-25nm at lower concentration and some of them intacted each other. It showed that the EAFP molecular interactions were so strong to form clusters spontaneously in aqueous solution. Eucommia antifungal protein(EAFP) crystals can be easily grown into big crystals in several hours.
2．By AFM the dynamic topographic changes were observed on the surfaces of several EAFP crystals and growth rates were measured at different supersaturations. The results indicated that growth rates of EAFP crystals were related to supersaturations. At higher supersaturation(σ=1.78) the EAFP crystals grew very fast; at moderate supersaturation(σ=1.5) the growth rates were 12 nm/s and 24.2 nm/s along the axes b, c of the {100} surface respectively; Even at lower supersaturation the EAFP crystals grew almost as fast as other protein crystals did. We concluded that this could account for the fast growth of EAFP crystals. In this article the effects of concentration of precipitator were presented, which were observed by AFM at lower supersaturation.
3．EAFP crystals didn’t grow exclusively by using a single mechanism, but by various growth modes corresponding to a certain protein concentrations. In addition to solid and stable features of EAFP molecules and compact stacking with little water, the growth features of EAFP monoclinic form crystals can be summarized as follows: ①Fast occurrence of the strong asymmetric multi-2D island stack growth sources; ②strong asymmetric spiral dislocation growth sources at low supersaturations; ③continuous generation of the step split during the process of growth;④growth by
extension of the troop of steps;All these maybe are related to the rapid growth of EAFP crystals.
4. At low-to-moderate supersaturations the single-double-like spiral dislocations dominated the growth on the {100} faces of the EAFP crystals. The source center was very anisotropic which was similar to the whole shape of the big crystal. So the center also controlled the shapes of crystals during the growth process. The source density was very low than those already reported before. The mean height of these steps was 1.9 nm which was equivalent to one unit of axis a. We have discussed the formation of single-double dislocation on the {100} face of EAFP crystals in more details. We found that during the growth process the double steps first grow separately and the connected by short bridge before tangential growth. After eighth or ninth layer the spiral steps would merge into intact elliptic layers. At the merging point right-handed spiral sources might appear but the left ones always overgrow it which made the whole growth topography looked like left-handed apparently. It indicated that the EAFP molecules has strong ability to adjust itself to adapt the environment.
5. Through various analysis of interactions in the EAFP crystals it indicated that there were 218 residue interactions between 12 symmetry-related EAFP molecule
本论文通过AFM实验主要进行了部分蛋白质晶体的生长过程的研究，包括杜仲抗真菌蛋白(Eucommia antifungal protein, 简称EAFP)、天花粉蛋白(Trichosanthin, 简称TCS)、天麻抗真菌蛋白(Gastrodia elata antifungal protein, 简称GAFP)，并取得了一定进展，有一些新的发现：
1．EAFP在其水溶液中能形成一种环状聚集体，在40mg/ml的高浓度时其直径大小从100nm到300nm左右， 而在低浓度(5mg/m/l)时只有10-25nm左右，且数量较少。有的形成一个完整的环，有的环正在形成。这说明EAFP分子间本身有着较强的相互作用，能自发地在水溶液中进行聚集，且随溶液的浓度变小而聚集能力降低。
2．EAFP晶体的生长速度很快，通常只需要数小时就可长到较大尺寸(约1mm)，要比普通的蛋白质晶体生长快很多倍， 而且沿着晶体学轴向的生长速率也各不相同，表现出明显的各向异性；且生长的速率还与蛋白质溶液的过饱和度密切相关，过饱和度较高时(大于1.78)生长极为快速，难以进行速率的测量；当过饱和度为1.45时, EAFP晶体的{100}面上的生长速率b方向上平均为12 nm/s, c方向上平均为24.2 nm/s；当过饱和度为1.15时, 其生长速率b方向上为4.1nm/s；c方向上为8.8nm/s, 而此时，其生长速率仍与报道的其他蛋白质晶体生长速率基本相当，这些都说明EAFP晶体具有很快的生长速率。
3．实时原位AFM研究EAFP单斜晶体{100}面的生长过程结果表明EAFP晶
体生长并非以单一的生长模式进行，而是随生长条件的变化采取不同的生长特征，有以下因素可能与EAFP的晶体快速生长机制相关：① 多重不对称二维岛堆垛生长源；② 活泼的不对称螺旋错位生长源(主要以不对称的双左旋为主，以不断出现的多个无规的右旋为次)；③ 台阶按编组生长的动态特性。
4．EAFP在低过饱和度下的螺旋生长过程中会出现台阶的合并，从而表现出单-双的螺旋台阶， 在小范围扫描中捕获到了这种现象，使得对螺旋生长台阶的合并过程有了更加深入的认识；并且台阶的生长受到沿晶体学b轴方向上的21螺旋对称的控制；当台阶生长至第八到第九层时，螺旋台阶会自动地形成完整的生长层，这充分说明了EAFP分子或分子簇具有良好的自身调节能力，这样在晶体生长过程中，晶体的缺陷减少，晶体质量提高，这也可说明EAFP晶体的x射线衍射能力很强的原因。
5．通过对EAFP晶体中各种相互作用分析表明：一个EAFP分子与周围的相邻分子间有218个残基间的相互作用，其中有18个是强的相互作用，有72个较强作用(间距小于4埃), 这些作用大部分与晶体学c方向上的作用相关, 说明在c方向上作用较强。由于这种差别，使得EAFP在结晶过程中，当分子受到不同方向上的作用力时，便形成了表面形貌的不对称，进而影响到晶体的外貌。另外，通过大分子结构分析XPLOR(NIH版)对EAFP在溶液中和在晶格中的分子能量的较为详尽的分析表明，尽管两种状态下的EAFP分子骨架差别不大(主要是侧链Arg残基伸展方向不同)，但晶格中的EAFP分子采用较为有利的构象，使结晶过程能够顺利进行；且沿结晶学不同方向上分子间的相互作用能（尤其是氢键能的差别）也不同，沿c方向上要比沿b方向上强约1/3，这也与AFM结果分析相一致。
6．发现天花粉晶体所有表面上没有螺旋位错或台阶生长，只在其{001}面上发现明显的非对称线性的3D核进行生长。这些线性聚体均沿着晶体学<110>姆较蚪信帕校蚁嗷テ叫校饪伤得魈旎ǚ鄯肿蛹湓谡飧龇较蛏嫌凶沤锨康南嗷プ饔茫沟梅肿友刈耪飧龇较蛉菀捉芯奂欢杂贕AFP晶体，原位实时动态的原子力显微成像初步的研究发现在其晶体的{001}表面晶体生长较为缓慢，没有发现台阶和螺旋位错生长现象，而是以分子聚集体的形式，慢慢从溶液中沉积到晶体表面，初步确定为法向生长(normal growth)。
由于蛋白质分子本身和蛋白质晶体生长体系的复杂性，本文的工作仅针对所研究对象进行讨论，进一步更深入的研究尚在进行中。
Biomolecules(e.g., proteins, nucleic acids and viruses) with specific structures play very important roles to many biological processes. To meet increasing demands of precise biomolecular 3-D structures,especially protein structures by X-ray diffraction, it is more and more necessary to study their crystal growth. However,growing protein crystals is very difficult and less knowledge of biomolecular crystal growth mechanism, process of nucleic formation, basic thermodynamic and kinetic parameters aren’t known as well as crystal defects, molecular orientable forces, etc. In order to learn their growth mechanisms, some technologies have been used such as Quasi-Elastic Light Scaterring(QELS) and Atomic Force Microscopy (AFM), and so far AFM is one of the most important tools. In this dissertation some protein crystal growth have mainly been studied by AFM such as Eucommia antifungal protein(EAFP), Trichosanthin(TCS), Gastrodia elata antifungal protein(TCS),especially on EAFP crystals.
1．In the solution EAFP molecules can form ring-like clusters(about 40-300nm) at higher concentration(40mg/ml) and about 10-25nm at lower concentration and some of them intacted each other. It showed that the EAFP molecular interactions were so strong to form clusters spontaneously in aqueous solution. Eucommia antifungal protein(EAFP) crystals can be easily grown into big crystals in several hours.
2．By AFM the dynamic topographic changes were observed on the surfaces of several EAFP crystals and growth rates were measured at different supersaturations. The results indicated that growth rates of EAFP crystals were related to supersaturations. At higher supersaturation(σ=1.78) the EAFP crystals grew very fast; at moderate supersaturation(σ=1.5) the growth rates were 12 nm/s and 24.2 nm/s along the axes b, c of the {100} surface respectively; Even at lower supersaturation the EAFP crystals grew almost as fast as other protein crystals did. We concluded that this could account for the fast growth of EAFP crystals. In this article the effects of concentration of precipitator were presented, which were observed by AFM at lower supersaturation.
3．EAFP crystals didn’t grow exclusively by using a single mechanism, but by various growth modes corresponding to a certain protein concentrations. In addition to solid and stable features of EAFP molecules and compact stacking with little water, the growth features of EAFP monoclinic form crystals can be summarized as follows: ①Fast occurrence of the strong asymmetric multi-2D island stack growth sources; ②strong asymmetric spiral dislocation growth sources at low supersaturations; ③continuous generation of the step split during the process of growth;④growth by
extension of the troop of steps;All these maybe are related to the rapid growth of EAFP crystals.
4. At low-to-moderate supersaturations the single-double-like spiral dislocations dominated the growth on the {100} faces of the EAFP crystals. The source center was very anisotropic which was similar to the whole shape of the big crystal. So the center also controlled the shapes of crystals during the growth process. The source density was very low than those already reported before. The mean height of these steps was 1.9 nm which was equivalent to one unit of axis a. We have discussed the formation of single-double dislocation on the {100} face of EAFP crystals in more details. We found that during the growth process the double steps first grow separately and the connected by short bridge before tangential growth. After eighth or ninth layer the spiral steps would merge into intact elliptic layers. At the merging point right-handed spiral sources might appear but the left ones always overgrow it which made the whole growth topography looked like left-handed apparently. It indicated that the EAFP molecules has strong ability to adjust itself to adapt the environment.
5. Through various analysis of interactions in the EAFP crystals it indicated that there were 218 residue interactions between 12 symmetry-related EAFP molecule
引文
[1] McPherson A. Crystallization of Biological Macromolecules. Cold Spring Harbor Laboratory Press, 1999.
[2] N.Feather. Historical Introduction to "X-rays and the Electric Conductivity of Gases". published by J J Thomson and Rutherford, Edinburg: E &S Livingstone LTD, 1958;p.6.
[3] W.C.Rontgen. On a New Kind of Rays-First Communication. The World of Atom 1966; Vol. I:390-396.
[4] 王大成主编，蛋白质工程. 北京: 化学工业出版社, 2002: 171-174.
[5] McPherson A. A brief history of protein crystal growth. J Cryst Growth 1991; 110:1.
[6] Bernal J.D., Fankuchen I., Riley D.P. Structure of the crystals of tomato bushy stunt virus preparations. nature 1938; 142:1075.
[7] Sumner J.B. The isolation and the crystallization of the enzyme urease. J Biol Chem 1926; 69:435.
[8] 王大成. 后基因组时代中的结构生物学. 生物化学与生物物理进展 2000; 27(4):340-344.
[9] J.Craig Venter, Mark D.Adams, et al. The Sequence of the Human Genome. Science 2001; 291:1304-1351.
[10] Jun Yu, Songnian Hu, et al. A Draft Sequence of the Rice Genome( Oryza sativa L. ssp. indica). Science 2002; 296:79-92.
[11] Takuji Sasaki, Takashi Matsumoto. The genome sequence and structure of rice chromosome 1. Nature 2002; 420:312-316.
[12] McPherson A. Macromolecular crystals. Sci Am 1989; 260:62.
[13] Malkin AJ, Kuznetsov Y, Land TA, DeYoreo JJ, McPherson A. Mechanisms of growth for protein and virus crystals. Nat Struct Biol 1995; 2(11):956-959.
[14] McPherson A, Malkin AJ, Kuznetsov YG. The science of macromolecular crystallization. Structure 1995; 3(8):759-768.
[15] Malkin AJ, Kuznetsov Y.G., Glantz W., McPherson A. Atomic force microscopy studies of surface morphology and growth kinetics in thaumatin crystallization. J Phys Chem 1996; 100:11736.
[16] Wood E.A. Crystals and light: An introduction to optical crystallography. Dover, New York 1977.
[17] Matthews B.W. Solvent content of protein crystals. J Mol Biol 1968; 33:491.
[18] Rose G.D., Wolfenden R. Hydrogen bonding, hydrophobicity, packing, and protein folding. Annu Rev Biophys Biomol Struct 1993; 22:381-415.
[19] Chernov A.A., Rashkovich L.N., Smoliskii I.L., Kuznetsov Y.G., et al. Growth of Crystals. In: EI.Givargizov and S.A.Grinberg, editor. New York: Consultant Bureau, 1988: p.43.
[20] Drenth J.and Haas C. Protein crystals and their stability. J Cryst Growth 1992; 122:107.
[21] Burton W.K., Cabrera N.and Frank F.C. The growth of crystals and the equilibrium structure of their surfaces. Philos Trans R Soc Lond 1951; A 243:299.
[22] Chernov A.A. Roughening and melting of crystalline surfaces. Prog Cryst Growth 1993; 26:195.
[23] Chernov A.A. Modern crystallization, Crystal Growth. Berlin: Springer-Verlag, 1984.
[24] Kuznetsov YG, Malkin A.J., Glantz W., McPherson A. In situ atomic force microscopy studies of protein and virus crystal growth mechanisms. J Cryst Growth 1996; 168:63-73.
[25] Malkin AJ, Land TA, Kuznetsov YG, McPherson A, DeYoreo JJ. Investigation of virus crystal growth mechanisms by in situ atomic force microscopy. PHYSICAL REVIEW LETTERS 1995; 75(14):2778-2781.
[26] Ng JD, Kuznetsov YG, Malkin AJ, Keith G, Giege R, McPherson A. Visualization of RNA crystal growth by atomic force microscopy. Nucleic Acids Res 1997; 25(13):2582-2588.
[27] Astier J.P., Bokern D., Lapena L., Veesler S. a-amylase crystal growth investigated by in situ atomic force microscopy. J Cryst Growth 2001; 226:294-302.
[28] Nakada T., Sazaki Gen, M Miyshita, Durbin S., Komatsu H. Direct AFM obervations of impurity effects on a lysozyme crystal. J Cryst Growth 1999; 196:503-510.
[29] Land T.A., Yoreo J.J.De. The evolution of growth modes and activity of growth sources on canavalin investigated by in situ atomic force microscopy. J Cryst Growth 2000; 208:623-637.
[30] Malkin AJ, Yu.G.Kuznetsov, McPherson A. An in situ AFM investigation of catalase crystallization. Surface Science 1997; 393:95-107.
[31] Jackson K.A. Present state of the theory of crystal growth from the melt. J Cryst Growth 1974; 24-25:130.
[32] Yu.G.Kuznetsov, A.J.Malkin, A.McPherson. The influence of precipitant concentration on macromolecular crystal growth mechanisms. J Cryst Growth 2001; 232:114-118.
[33] DeLucas L.J., Smith C.D., Smith H.W., Vijaykumar S. Protein crystal growth in microgravity. Science 1989; 246:651-654.
[34] Giege R., Drenth J., Ducriux A., McPherson A., and Saenger W. Crystallogenesis of biological macromolecules-Biological, microgravity and other physicochemical aspects. Prog Crystal growth Characterization Mater 1995; 30:237-281.
[35] Van der Klei I.J., Lawson C.L., Rozeboom H., Dijkstra B.W., et al. Use of electron microscopy in the examination of lattice defects in crystals of alcohol oxidase. FEBS Lett 1989; 244:213.
[36] Sato K., Fukuba Y., Mitsuda T., Hirai K., and Moriya K. Observation of lattice defects in orthorhombic hen-egg white lysozyme crystals with laser scattering tomography. J Cryst Growth 1992; 122:87.
[37] Malkin AJ, Kuznetsov YG, McPherson A. Defect Structure of Macromolecular Crystals. J Structure Biology 1996; 117:124-137.
[38] Koszelak S., Day J., Leja C., Cudney R., and McPherson. Protein and virus crystal growth on international microgravity laboratory-2. Biophys J 1995; 69:13.
[39] McPherson A., Spencer R. Preliminary structure analysis of canavalin from jack bean. Arch Biochem Biophys 1975; 169:650.
[40] Ko TP, Ng JD, McPherson A. The three dimensional structure of canavalin from jack bean(Canavalia ensiformis). Plant Physiol 1993; 101:729.
[41] Ko TP, Day J., Greenwood A, McPherson A. The structures of three crystal of forms of the sweet protein thaumatin. Acta Crystallogr D 1994; 50:813.
[42] Ko TP, Ng JD, Day J., Greenwood A, McPherson A. X-ray structure determination of three crystal forms of canavalin by molecular replacement. Acta Crystallogr D 1993; 49:478.
[43] Binnig G., Quate C.F., Gerber C. Atomic force microscope. Phys Rev Lett 1986; 56:930-933.
[44] Durbin S.D., Carlson W.E. Lysozyme crystal growth studied by atomic force microscopy. J Cryst Growth 1992; 122:71-79.
[45] Durbin S.D., Carlson W.E., Saros M.T. In situ studies of protein crystallization by atomic force microscopy. J Phys D: Appl Phys 1993; 26:128-132.
[46] Yip CM, DeFelippis MR, Frank BH, Brader ML, Ward MD. Structural and morphological characterization of ultralente insulin crystals by atomic force microscopy: evidence of hydrophobically driven assembly. Biophys J 1998; 75(3):1172-1179.
[47] Yip CM, Brader ML, DeFelippis MR, Ward MD. Atomic force microscopy of crystalline insulins:the influence of sequence variation on crystallization and interfacial structure. Biophys J 1998; 74(5):2199-2208.
[48] Yip CM, Ward MD. Atomic force microscopy of insulin single crystals-direct visualization of molecules and crystal growth. Biophys J 1998; 71:1071-1078.
[49] Land TA, Malkin AJ, Kuznetsov YG, McPherson A, De Yoreo JJ. Mechanisms of protein crystal growth: An atomic force microscopy study of canavalin crystallization. PHYSICAL REVIEW LETTERS 1995; 75(14):2774-2777.
[50] Yau S.T., Thomas B.R., Vekilov P.G. Molecular mechanisms of cystallization and defect
formation. Physical Review Letters 2000; 85:353-356.
[51] Li M.R., Nadarajah A., Pusey M.L. Growth of (101) faces of tetragonal lysozyme crystals:determination of the growth mechanism. Acta Cryst 1999; D 55:1012-1022.
[52] Li H.Y., Perozzo M.A., Konnert J.H., Nadarajah A., Pusey M.L. Determining the molecular-packing arrangements on protein crystal faces by atomic force microscopy. Acta Cryst 1999; D 55:1023-1035.
[53] Li M.R., Nadarajah A., Pusey M.L. Determining the molecular growth mechanisms of protein crystal faces by atomic force microscopy. Acta Cryst 1999; D55:1036-1045.
[54] Malkin AJ, Kuznetsov YG, McPherson A. Incorporation of microcrystals by growing protein and virus crystals. Proteins: Structure, Function and Genetics 1996; 24(2):247-252.
[55] Kuznetsov Yu.G., Konnert J., Malkin A.J., McPherson A. The advancement and structure of growth steps on thaumatin crystals visualized by atomic force microscopy at molecular resolution. Surface Science 1999; 440:69-80.
[56] Kuznetsov YG, Malkin AJ, Land TA, DeYoreo JJ, Barba AP, Konnert J, McPherson A. Molecular resolution imaging of macromolecular crystals by atomic force microscopy. Biophys J 1997; 72(5):2357-2364.
[57] Plomp M., McPherson A, Larson S.B., Malkin A.J. Growth Mechanisms and Kinetics of Trypsin Crystallization. Physical Chem 2001; 105(2):542-551.
[58] Kuznetsov Yu.G., Malkin A.J., McPherson A. AFM studies of the nucleation and growth mechanisms of macromolecular crystals. J Cryst Growth 1999; 196:489-502.
[59] Kuznetsov Y.G., Malkin A.J., Lucas RW, Plomp M., McPherson A. Imaging of viruses by atomic force microscopy. J Gen Virol 2001; 82:2025-2034.
[60] Williams T. Invertebrate iridescent viruses. New York: Plenum Press, 1998.
[61] McPherson A. The preparation and Analysis of Protein Crystals. New York: Wiley & Sons, 1982.
[62] Weber P.C. Overview of protein crystallization methods:macromolecular crystallography. Methods Enzymol 1997; 276A.
[63] Chernov A.A., Komatsu H. Topics in crystal growth kinetics. In science and Technology of Crystal Growth. In: JP vander Eerden, editor. The Netherlands Kluwer, 1995: 67-79.
[64] Buckley H.E. Crystal Growth. London: Wiley & Sons, 1951.
[65] 刘小烛,胡忠,李英等. 杜仲皮中抗真菌蛋白的分离和特性研究，杜仲皮中抗真菌蛋白的分离和特性研究，4,1994.
[66] Xiang Y, Huang R, Liu W, et al. Crystallization and Preliminary crystallographic studies of a novel protein with five disulfide bridges from Eucommia ulmoides Oliver. Acta Cryst, 2002,
D58: 1838-1840.
[67] Huang R, Xang Y, Liu X, et al. Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv. FEBS Letters, 2002, 521: 87-90.
[68] 汪盛，向烨，李根培，王大成. 杜仲抗真菌蛋白(EAFP)晶体生长的AFM研究——快速生长与晶面生长速率, 生物化学与生物物理进展，30(5):784-791(2003).
[69] Rong L, Yamane T, Niimura N. Measurement and control of the crystal growth rate of tetragonal hen egg-white lysozyme imaged with an atomic force microscope. J. Cryst. Growth, 2000, 217: 161-169.
[70] McPherson A, Crystallization of Biological Macromolecules 1999, COLD SPRING HARBOR LABORATORY PRESS. Cold Spring Harbor, New York., pp 398-401.
[71] Chernov A A. Crystal Growth and Crystallography. Acta Cryst, 1998, A54: 859-872.
[72] Rossmann, M.G. & van Beek, C.G. (1999), "Data processing", Acta Cryst. D55, 1631-1653.
[73] Collaborative Computational Project, Number 4. The CCP4 Suite: Programs for Protein Crystallography，Acta Cryst. 1994，D50, 760-763.
[74] McRee, D.E. "XtalView/Xfit - A Versatile Program for Manipulating Atomic Coordinates and Electron Density".? J. Structural Biology, 1999，125, 156-165.
[75] Per J. Kraulis MOLSCRIPT: A Program to Produce Both Detailed and Schematic Plots of Protein Structures. Journal of Applied Crystallography，1991，vol 24, pp 946-950.
[76] Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S. & Dodson, E. J. Acta Cryst. D55, 1999，247-2555.
[77] Sheldrick, G. M. (1997). SHELX97. University of Go ttingen, Germany.
[78] W. Liu, Y.-L. Hu, M. Wang, et al. Purification, crystallization and preliminary X-ray diffraction analysis of a novel mannose-binding lectin from Gastrodia elata with antifungal properties, Acta Cryst. (2002). D58, 1833-1835.
[2] N.Feather. Historical Introduction to "X-rays and the Electric Conductivity of Gases". published by J J Thomson and Rutherford, Edinburg: E &S Livingstone LTD, 1958;p.6.
[3] W.C.Rontgen. On a New Kind of Rays-First Communication. The World of Atom 1966; Vol. I:390-396.
[4] 王大成主编，蛋白质工程. 北京: 化学工业出版社, 2002: 171-174.
[5] McPherson A. A brief history of protein crystal growth. J Cryst Growth 1991; 110:1.
[6] Bernal J.D., Fankuchen I., Riley D.P. Structure of the crystals of tomato bushy stunt virus preparations. nature 1938; 142:1075.
[7] Sumner J.B. The isolation and the crystallization of the enzyme urease. J Biol Chem 1926; 69:435.
[8] 王大成. 后基因组时代中的结构生物学. 生物化学与生物物理进展 2000; 27(4):340-344.
[9] J.Craig Venter, Mark D.Adams, et al. The Sequence of the Human Genome. Science 2001; 291:1304-1351.
[10] Jun Yu, Songnian Hu, et al. A Draft Sequence of the Rice Genome( Oryza sativa L. ssp. indica). Science 2002; 296:79-92.
[11] Takuji Sasaki, Takashi Matsumoto. The genome sequence and structure of rice chromosome 1. Nature 2002; 420:312-316.
[12] McPherson A. Macromolecular crystals. Sci Am 1989; 260:62.
[13] Malkin AJ, Kuznetsov Y, Land TA, DeYoreo JJ, McPherson A. Mechanisms of growth for protein and virus crystals. Nat Struct Biol 1995; 2(11):956-959.
[14] McPherson A, Malkin AJ, Kuznetsov YG. The science of macromolecular crystallization. Structure 1995; 3(8):759-768.
[15] Malkin AJ, Kuznetsov Y.G., Glantz W., McPherson A. Atomic force microscopy studies of surface morphology and growth kinetics in thaumatin crystallization. J Phys Chem 1996; 100:11736.
[16] Wood E.A. Crystals and light: An introduction to optical crystallography. Dover, New York 1977.
[17] Matthews B.W. Solvent content of protein crystals. J Mol Biol 1968; 33:491.
[18] Rose G.D., Wolfenden R. Hydrogen bonding, hydrophobicity, packing, and protein folding. Annu Rev Biophys Biomol Struct 1993; 22:381-415.
[19] Chernov A.A., Rashkovich L.N., Smoliskii I.L., Kuznetsov Y.G., et al. Growth of Crystals. In: EI.Givargizov and S.A.Grinberg, editor. New York: Consultant Bureau, 1988: p.43.
[20] Drenth J.and Haas C. Protein crystals and their stability. J Cryst Growth 1992; 122:107.
[21] Burton W.K., Cabrera N.and Frank F.C. The growth of crystals and the equilibrium structure of their surfaces. Philos Trans R Soc Lond 1951; A 243:299.
[22] Chernov A.A. Roughening and melting of crystalline surfaces. Prog Cryst Growth 1993; 26:195.
[23] Chernov A.A. Modern crystallization, Crystal Growth. Berlin: Springer-Verlag, 1984.
[24] Kuznetsov YG, Malkin A.J., Glantz W., McPherson A. In situ atomic force microscopy studies of protein and virus crystal growth mechanisms. J Cryst Growth 1996; 168:63-73.
[25] Malkin AJ, Land TA, Kuznetsov YG, McPherson A, DeYoreo JJ. Investigation of virus crystal growth mechanisms by in situ atomic force microscopy. PHYSICAL REVIEW LETTERS 1995; 75(14):2778-2781.
[26] Ng JD, Kuznetsov YG, Malkin AJ, Keith G, Giege R, McPherson A. Visualization of RNA crystal growth by atomic force microscopy. Nucleic Acids Res 1997; 25(13):2582-2588.
[27] Astier J.P., Bokern D., Lapena L., Veesler S. a-amylase crystal growth investigated by in situ atomic force microscopy. J Cryst Growth 2001; 226:294-302.
[28] Nakada T., Sazaki Gen, M Miyshita, Durbin S., Komatsu H. Direct AFM obervations of impurity effects on a lysozyme crystal. J Cryst Growth 1999; 196:503-510.
[29] Land T.A., Yoreo J.J.De. The evolution of growth modes and activity of growth sources on canavalin investigated by in situ atomic force microscopy. J Cryst Growth 2000; 208:623-637.
[30] Malkin AJ, Yu.G.Kuznetsov, McPherson A. An in situ AFM investigation of catalase crystallization. Surface Science 1997; 393:95-107.
[31] Jackson K.A. Present state of the theory of crystal growth from the melt. J Cryst Growth 1974; 24-25:130.
[32] Yu.G.Kuznetsov, A.J.Malkin, A.McPherson. The influence of precipitant concentration on macromolecular crystal growth mechanisms. J Cryst Growth 2001; 232:114-118.
[33] DeLucas L.J., Smith C.D., Smith H.W., Vijaykumar S. Protein crystal growth in microgravity. Science 1989; 246:651-654.
[34] Giege R., Drenth J., Ducriux A., McPherson A., and Saenger W. Crystallogenesis of biological macromolecules-Biological, microgravity and other physicochemical aspects. Prog Crystal growth Characterization Mater 1995; 30:237-281.
[35] Van der Klei I.J., Lawson C.L., Rozeboom H., Dijkstra B.W., et al. Use of electron microscopy in the examination of lattice defects in crystals of alcohol oxidase. FEBS Lett 1989; 244:213.
[36] Sato K., Fukuba Y., Mitsuda T., Hirai K., and Moriya K. Observation of lattice defects in orthorhombic hen-egg white lysozyme crystals with laser scattering tomography. J Cryst Growth 1992; 122:87.
[37] Malkin AJ, Kuznetsov YG, McPherson A. Defect Structure of Macromolecular Crystals. J Structure Biology 1996; 117:124-137.
[38] Koszelak S., Day J., Leja C., Cudney R., and McPherson. Protein and virus crystal growth on international microgravity laboratory-2. Biophys J 1995; 69:13.
[39] McPherson A., Spencer R. Preliminary structure analysis of canavalin from jack bean. Arch Biochem Biophys 1975; 169:650.
[40] Ko TP, Ng JD, McPherson A. The three dimensional structure of canavalin from jack bean(Canavalia ensiformis). Plant Physiol 1993; 101:729.
[41] Ko TP, Day J., Greenwood A, McPherson A. The structures of three crystal of forms of the sweet protein thaumatin. Acta Crystallogr D 1994; 50:813.
[42] Ko TP, Ng JD, Day J., Greenwood A, McPherson A. X-ray structure determination of three crystal forms of canavalin by molecular replacement. Acta Crystallogr D 1993; 49:478.
[43] Binnig G., Quate C.F., Gerber C. Atomic force microscope. Phys Rev Lett 1986; 56:930-933.
[44] Durbin S.D., Carlson W.E. Lysozyme crystal growth studied by atomic force microscopy. J Cryst Growth 1992; 122:71-79.
[45] Durbin S.D., Carlson W.E., Saros M.T. In situ studies of protein crystallization by atomic force microscopy. J Phys D: Appl Phys 1993; 26:128-132.
[46] Yip CM, DeFelippis MR, Frank BH, Brader ML, Ward MD. Structural and morphological characterization of ultralente insulin crystals by atomic force microscopy: evidence of hydrophobically driven assembly. Biophys J 1998; 75(3):1172-1179.
[47] Yip CM, Brader ML, DeFelippis MR, Ward MD. Atomic force microscopy of crystalline insulins:the influence of sequence variation on crystallization and interfacial structure. Biophys J 1998; 74(5):2199-2208.
[48] Yip CM, Ward MD. Atomic force microscopy of insulin single crystals-direct visualization of molecules and crystal growth. Biophys J 1998; 71:1071-1078.
[49] Land TA, Malkin AJ, Kuznetsov YG, McPherson A, De Yoreo JJ. Mechanisms of protein crystal growth: An atomic force microscopy study of canavalin crystallization. PHYSICAL REVIEW LETTERS 1995; 75(14):2774-2777.
[50] Yau S.T., Thomas B.R., Vekilov P.G. Molecular mechanisms of cystallization and defect
formation. Physical Review Letters 2000; 85:353-356.
[51] Li M.R., Nadarajah A., Pusey M.L. Growth of (101) faces of tetragonal lysozyme crystals:determination of the growth mechanism. Acta Cryst 1999; D 55:1012-1022.
[52] Li H.Y., Perozzo M.A., Konnert J.H., Nadarajah A., Pusey M.L. Determining the molecular-packing arrangements on protein crystal faces by atomic force microscopy. Acta Cryst 1999; D 55:1023-1035.
[53] Li M.R., Nadarajah A., Pusey M.L. Determining the molecular growth mechanisms of protein crystal faces by atomic force microscopy. Acta Cryst 1999; D55:1036-1045.
[54] Malkin AJ, Kuznetsov YG, McPherson A. Incorporation of microcrystals by growing protein and virus crystals. Proteins: Structure, Function and Genetics 1996; 24(2):247-252.
[55] Kuznetsov Yu.G., Konnert J., Malkin A.J., McPherson A. The advancement and structure of growth steps on thaumatin crystals visualized by atomic force microscopy at molecular resolution. Surface Science 1999; 440:69-80.
[56] Kuznetsov YG, Malkin AJ, Land TA, DeYoreo JJ, Barba AP, Konnert J, McPherson A. Molecular resolution imaging of macromolecular crystals by atomic force microscopy. Biophys J 1997; 72(5):2357-2364.
[57] Plomp M., McPherson A, Larson S.B., Malkin A.J. Growth Mechanisms and Kinetics of Trypsin Crystallization. Physical Chem 2001; 105(2):542-551.
[58] Kuznetsov Yu.G., Malkin A.J., McPherson A. AFM studies of the nucleation and growth mechanisms of macromolecular crystals. J Cryst Growth 1999; 196:489-502.
[59] Kuznetsov Y.G., Malkin A.J., Lucas RW, Plomp M., McPherson A. Imaging of viruses by atomic force microscopy. J Gen Virol 2001; 82:2025-2034.
[60] Williams T. Invertebrate iridescent viruses. New York: Plenum Press, 1998.
[61] McPherson A. The preparation and Analysis of Protein Crystals. New York: Wiley & Sons, 1982.
[62] Weber P.C. Overview of protein crystallization methods:macromolecular crystallography. Methods Enzymol 1997; 276A.
[63] Chernov A.A., Komatsu H. Topics in crystal growth kinetics. In science and Technology of Crystal Growth. In: JP vander Eerden, editor. The Netherlands Kluwer, 1995: 67-79.
[64] Buckley H.E. Crystal Growth. London: Wiley & Sons, 1951.
[65] 刘小烛,胡忠,李英等. 杜仲皮中抗真菌蛋白的分离和特性研究，杜仲皮中抗真菌蛋白的分离和特性研究，4,1994.
[66] Xiang Y, Huang R, Liu W, et al. Crystallization and Preliminary crystallographic studies of a novel protein with five disulfide bridges from Eucommia ulmoides Oliver. Acta Cryst, 2002,
D58: 1838-1840.
[67] Huang R, Xang Y, Liu X, et al. Two novel antifungal peptides distinct with a five-disulfide motif from the bark of Eucommia ulmoides Oliv. FEBS Letters, 2002, 521: 87-90.
[68] 汪盛，向烨，李根培，王大成. 杜仲抗真菌蛋白(EAFP)晶体生长的AFM研究——快速生长与晶面生长速率, 生物化学与生物物理进展，30(5):784-791(2003).
[69] Rong L, Yamane T, Niimura N. Measurement and control of the crystal growth rate of tetragonal hen egg-white lysozyme imaged with an atomic force microscope. J. Cryst. Growth, 2000, 217: 161-169.
[70] McPherson A, Crystallization of Biological Macromolecules 1999, COLD SPRING HARBOR LABORATORY PRESS. Cold Spring Harbor, New York., pp 398-401.
[71] Chernov A A. Crystal Growth and Crystallography. Acta Cryst, 1998, A54: 859-872.
[72] Rossmann, M.G. & van Beek, C.G. (1999), "Data processing", Acta Cryst. D55, 1631-1653.
[73] Collaborative Computational Project, Number 4. The CCP4 Suite: Programs for Protein Crystallography，Acta Cryst. 1994，D50, 760-763.
[74] McRee, D.E. "XtalView/Xfit - A Versatile Program for Manipulating Atomic Coordinates and Electron Density".? J. Structural Biology, 1999，125, 156-165.
[75] Per J. Kraulis MOLSCRIPT: A Program to Produce Both Detailed and Schematic Plots of Protein Structures. Journal of Applied Crystallography，1991，vol 24, pp 946-950.
[76] Murshudov, G. N., Vagin, A. A., Lebedev, A., Wilson, K. S. & Dodson, E. J. Acta Cryst. D55, 1999，247-2555.
[77] Sheldrick, G. M. (1997). SHELX97. University of Go ttingen, Germany.
[78] W. Liu, Y.-L. Hu, M. Wang, et al. Purification, crystallization and preliminary X-ray diffraction analysis of a novel mannose-binding lectin from Gastrodia elata with antifungal properties, Acta Cryst. (2002). D58, 1833-1835.