生物物理学与冷冻电镜 王宏伟 生物新馆320 hongweiwang 什么是生物物理学？ 生物物理学是运用物理学的理论、技 术和方法，研究生命物质的物理性质 、生命过程的物理和物理化学规律， 以及物理因素对生物系统作用机制的 科学。 -互动百科 一个生物个体的物理学参数： 身高，体重，胸围，头围，年龄，。 变化 蛋白质分子的物理学参数： 分子量，形状，位置，运动速度，浓度，。 结构变化 Why do the parameters matter? Molecular Weight 分子量 (MW) 质谱（Mass Spectrometry） 测定分子量 Why do the parameters matter? Molecular Weight 分子量 (MW) Why do the parameters matter? 位置，运动与变化的速度，能量 显微镜 波谱学 二者的结合 各种光学显微镜照片 光谱 Absorption Control No blue/green light red filter B & G absorbed 光吸收 1993-2010 J.Paul Robinson - Purdue University Cytometry Laboratories white lightblue lightred lightgreen light B & G absorbed R & G absorbedB & R absorbed 1993-2010 J.Paul Robinson - Purdue University Cytometry Laboratories 光吸收 Ix I0 I x c M, x cm, cm2 mol-1 光吸收率测量浓度 荧光光谱学与荧光显微成像 吸收 荧光(Fluorescence) 吸收发射发射 荧光光谱仪 Absorption Spectrum The fluorescence emission spectrum is normally measured at a direction perpendicular to the incident light path to avoid the contamination from the strong incident light signals. 荧光光谱测量底物的结合 Collisional quenching is used extensively to probe relative burial of tryptophan residues in proteins. Commonly used quenchers for Trp fluorescence are iodide and acrylamide. Trp 荧光共振能量转移 (Fluorescence Resonance Energy Transfer) FRET 吸收发射 吸收发射 FRET信号强度与分子间距离相关 * * A molecular ruler E 1/R6 means that donor and acceptor must be close in space (in general, 100 or 10 nm) 单分子探测技术 The lac repressor LacI dissociates from the lac operator in response to inducer (top) and rebinds after dilution of the inducer (bottom ). By tracking spots carefully, find that LacI first binds non- specifically to DNA, then diffuses along DNA, occasionally jumping to different DNA segments. In searching for the operator, LacI spends 90% of its time nonspecifically bound to and diffusing along DNA with a residence time of 5 ms. Why do the parameters matter? Structure 结构 Seeing is believing 眼见为实 X-ray crystallography 晶体学 NMR spectroscopy 核磁共振波谱学 Cryo-electron microscopy 冷冻电子显微学 Electron micrograph of a thin section: 10 nm Fig. 1-11 Scratch of the idea of TEM (March 9, 1931) First TEM by Ernst Ruska (1933) From the Nobel Lecture by Ernst Ruska From Wikipedia Ernst Ruska (1906 1988) Nobel Prize in Physics 1986 “for his fundamental work in electron optics, and for the design of the first electron microscope” 透射电子显微镜的发明 自然 科学 超级显微镜对植物病毒的观察 人类第一次看到病毒分子 Helmant Ruska (1908 1973) The first structural information about a virus. TMV is a filamentous virus! Ernst Ruska, 1933JEOL 300 KV microscope HITACHI 1 MV microscope 电子显微镜 单颗粒电子显微学 SampleNegative stain solution Carbon grid Negatively stained specimen Put the specimen into an electron microscope Take magnified micrographs Thin carbon Image analysis and three dimensional reconstruction Grid ethane liquid nitrogen grid forceps Helen Saibil 冷冻电镜技术 Cryo Electron Microscopy Aquareovirus Particles Zhang et al., 2010, Cell 冷冻电镜可以提供原子分辨率的结构信息 冷冻电镜可以揭示近自然状态的结构信息 Pre-powerstroke (ADP-Pi) Post-powerstroke (nucleotide free) 肌球蛋白分子在肌动蛋白微丝上的运动 Dominguez et al., 1998, Cell Ron Vale Walker et al., 2000, Nature 噬菌体侵染细菌的过程 细菌的鞭毛运动机制 Yonekura K et al., 2003 Cryo-Tomography of Whole Cells Murphy et al., 2006, Nature 20 individual flagellar motor sub- tomograms were aligned, averaged, and symmetrized. Tomography of Treponema primitia whole cell embedded in ice. From Jensens Lab at Cal Tech 细菌中磁性小体的分布 什么是生物物理学？ 生物物理学通过对生物体或者生物分子的直接物 理参数的测量来揭示生命现象的内在规律。 Direct 生物物理学始终处于技术上的最前沿。 Cool 生物物理学通常提供精彩的图像和模型。 Fancy 生物物理与冷冻电镜 王宏伟 生物新馆320 hongweiwang The Inner Life of The Cell