纳米技术与生物学研究方法——冷冻电镜.ppt

1、2015.12.8 邢福临,Nanotechnology & biology,Content,Introduction Detection of biological nanoparticles,Introduction,10 3 ,10 6 ,10 9 ,10 10 ,1mm,1m,1nm, ,Introduction,Nano-biomaterial,Nano biological apparatus,Nano-biotechnology in clinic,Detection of biologicalparticles,primary structure,secondary struc

2、ture,tertiary structure,quaternary structure,Detection of biologicalparticles,X-ray diffraction,Wavelengh0.01-100, 0,Purification of protein Crystallization Data collection Modeling and revision,1953,Detection of biologicalparticles,X-ray diffraction,High resolution Non-destructivetosamples No pollu

3、tion Informative,Static conformation only HARD to crystallize!,Detection of biologicalparticles,Cryo-electron microscopy (cryo-EM),electron microscope electrons serve as the source of radiation,Electrons are accelerated down the microscope column at accelerating voltages of typically 80300 kV. Passi

4、ng through the specimen Scattered electrons are focused by the electromagnetic lenses of the microscope,Detection of biologicalparticles,Cryo-electron microscopy (cryo-EM),Fixation （osmic acid/ glutaraldehyde ） dehydration Staining (heavy metalsalt) Fltra-thinsections preparation,the lower electron

5、a poor signal-to-noise ratio,electron microscope,Detection of biologicalparticles,Cryo-electron microscopy (cryo-EM),Mitochondria &endoplasmicreticulum,chloroplast,electron microscope,Detection of biologicalparticles,Cryo-electron microscopy (cryo-EM),Imaging at liquid nitrogen temperatures reduces

6、the extent of radiation damage by as much as sixfold compared to ambient temperatures This means that for images recorded at cryogenic temperatures, higher electron doses can be used to increase the signal to noise ratio.,How to increase the signal-to-noise ratio?,Detection of biologicalparticles,Cr

7、yo-electron microscopy (cryo-EM),each image taken at a different tilt relative to the direction of the incident electron beam,These images may then be combined computationally, using a strategy similar to that used in computerized axial tomography,Detection of biologicalparticles,Cryo-electron micro

8、scopy (cryo-EM),Principle of reconstruction of 3D structure by Fourier inversion,projection images of the object, each with a different orientation, have 2D Fourier transforms that correspond to sections (indicated by red arrows) through the 3D Fourier transform of the original object. Once the 3D F

9、ourier transform is built up from a collection of 2D images spanning a complete range of orientations, Fourier inversion enables recovery of the 3D structure.,Detection of biologicalparticles,Cryo-electron microscopy (cryo-EM),biological specimens rapidly freezed in a layer of glass-like ice imaged

10、at liquid nitrogen and/or helium temperature averaging multiple images collected from frozen-hydrated specimens,Differences between EM and cryo-EM,Detection of biologicalparticles,Cryo-electron microscopy (cryo-EM),GroEL protein complexes,Detection of biologicalparticles,Cryo-electron microscopy (cr

11、yo-EM),GroEL protein complexes at approximately 7A resolution using approximately 28000 individual projection images,Detection of biologicalparticles,Cryo-electron microscopy (cryo-EM),Detection of biologicalparticles,Cryo-electron microscopy (cryo-EM),references,1郭强. 基于冷冻电镜的原核生物核糖体小亚基组装过程的研究.清华大学,2014. 2.Milne J L S, Borgnia M J, Bartesaghi A, et al. Cryoelectron microscopya primer for the nonmicroscopist. FEBS Journal, 2013, 280(1): 28-45. 3.Yan C, Hang J, Wan R, et al. Structure of a yeast spliceosome