生物物理学与冷冻电镜

1、生物物理学与冷冻电镜生物物理学与冷冻电镜王宏伟生物新馆什么是生物物理学？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

2、 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各种光学显微镜照片光谱光谱AbsorptionControlNo blue/green light red filterB & G absorbed光吸收光吸收 1993-2010 J.Paul Robinson - Purdue University Cytometry Laboratorieswhite lightblue lightred lightgreen lightB & G absorbedR & G absorbedB & R abso

3、rbed 1993-2010 J.Paul Robinson - Purdue University Cytometry Laboratories光吸收光吸收IxI0IxA cxc M, x cm, cm2 mol-1光吸收率测量浓度光吸收率测量浓度荧光光谱学与荧光显微成像荧光光谱学与荧光显微成像吸收荧光荧光(Fluorescence)(Fluorescence)吸收发射发射荧光光谱仪荧光光谱仪Absorption SpectrumThe fluorescence emission spectrum is normally measured at a direction perpendicul

4、ar 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荧光共振能量转移荧光共振能量转移

5、(Fluorescence Resonance Energy Transfer)FRET吸收发射吸收发射FRETFRET信号强度与分子间距离相关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)单分子探测技术单分子探测技术 The lac repressor LacI dissociates from the lac operator in response to inducer (top) and rebind

6、s 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

7、 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 nmFig. 1-11Scratch of the idea of TEM (March 9, 1931) First TEM by Ernst Rus

8、ka (1933)From the Nobel Lecture by Ernst RuskaFrom WikipediaErnst 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人类第一次看到病毒分子Helmant Ruska(1908 1973)The firs

9、t structural information about a virus.TMV is a filamentous virus!Ernst Ruska, 1933JEOL 300 KV microscopeHITACHI 1 MV microscope电子显微镜电子显微镜单颗粒电子显微学单颗粒电子显微学SampleNegative stain solutionCarbon gridNegatively stained specimenPut the specimen into an electron microscope Take magnified micrographsThin car

10、bonImage analysis and three dimensional reconstructionGridethaneliquid nitrogengridforcepsHelen Saibil冷冻电镜技术冷冻电镜技术 Cryo Electron MicroscopyAquareovirus ParticlesZhang 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冷冻电镜可以揭示近自然状态的结构信息Pre-powerstroke (ADP-Pi)Post-powerstroke(nu

11、cleotide free)肌球蛋白分子在肌动蛋白微丝上的运动肌球蛋白分子在肌动蛋白微丝上的运动Dominguez et al., 1998, CellRon ValeWalker et al., 2000, Nature噬菌体侵染细菌的过程噬菌体侵染细菌的过程细菌的鞭毛运动机制细菌的鞭毛运动机制Yonekura K et al., 2003Cryo-Tomography of Whole CellsMurphy et al., 2006, Nature20 individual flagellar motor sub-tomograms were aligned, averaged, and symmetrized.Tomography of Treponema primitia whole cell embedded in ice.From Jensens Lab at C