Lecture CIMST Winter School. 1. What can you see by TEM?

Transcription

1 Lecture CIMST Winter School Cryo-electron microscopy and tomography of biological macromolecules :00-9:45 in Y03G91 Dr. Takashi Ishikawa OFLB/005 Tel: Lab webpage: 1. What can you see by TEM? Transmission electron microscopy (TEM) is an imaging technique. It can visualize biological, organic and inorganic materials as far as the specimen is thin enough for electrons to go through. Here in this lecture, we will focus on TEM technology to observe 3D structure of biological macromolecules. 2. Hardware Diagram of the transmission electron microscope is similar to that of a light microscope (LM). Both of them have a gun. Electrons or photons are emitted from the gun, penetrate the specimen, focused and enlarged by lenses (TEM uses electromagnetic lenses) and projected on a screen or a camera. Inside of the electron microscope is vacuum. 3. Image formation by TEM In principle, micrographs of TEM are 2D projections of the 3D objects. However, there are significant differences due to weak interaction between the electron beam and the specimen (i.e. TEM specimen is transparent against electrons). Therefore, instead of amplitude contrast LM utilizes, image formation of TEM depends on phase contrast which utilizes interference of electron waves. For phase contrast image must not be perfectly focused; there must be defocus. 4. Specimen preparation Since the contrast between biological materials (density ~1.4) and water (1.0) is poor, previously templates of heavy metal were made to enhance the contrast. With these methodologies (shadowing and negative stain) only the surface of the molecules is visualized. In 1980 s cryo-em was developed. There ice-embedded biological macromolecules are observed directly at the liquid nitrogen temperature. 5. Resolution and radiation damage

2 Available discussion on the molecular structure depends on the level of resolution. Electron beams damage the specimen and limit the resolution. However, if the illumination is too dark, enough amount of contrast for image analysis cannot be obtained. The most essential and serious problem of 3D EM of biological molecules is this dilemma. 6. Various ways of three-dimensional reconstruction technique Here we will discuss four different approaches to reconstruct 3D structure of biological macromolecules from cryo-electron micrographs D crystal In the 2D crystal molecules array two-dimensionally. The Fourier transform of the image is a lattice. Thus, in the Fourier space, you can earn higher signal-to-noise ratio. Which enable the high quality data from lower illumination (i.e. lower radiation damage), resulting higher resolution (more than ten structures have been solved at the atomic resolution). However, this methodology demands, as X-ray crystallography, crystallization. Advantage, comparing to X-ray crystallography, is that we do not need heavy atoms, because direct images are recorded. For 3D information, tilted 2D crystals are inserted into the microscope with special holder. Since you cannot tilt 2D crystal at 90 degrees in the microscope, you will miss some information in the 3D Fourier space (missing cone). Example: bacteriorhodopsin, water channel Tubular crystal Molecules are arrayed as spirals. Since the signal will be spread along one dimensional lines (layer lines), the resolution is not so high as 2D crystals. All the necessary view angles for 3D reconstruction can be obtained from tubular crystal. Therefore, there is no missing information and tilt is not needed. Example: acetylcholine receptor, actin, microtubule 6-3. Single particle analysis This is a technique to calculate the 3D structure from many single particles (>5,000), determining their angles by computation. Since the molecules are dispersed in the solution, you can set solution condition freely, which allows you to see different stages of reaction, for example. However, the angle determination is complicated. Initially, you do not know either the final 3D structure or the view angles of individual particles. You must determine these two unknown factors at the same time. One approach is shown here: projection matching, which starts from a certain template and find the best-fitted view angle for each raw data particle. To generate initial templates, there are a few options. The resolution is evaluated using Fourier shell correlation. Example: Clp protease, ribosome 6-4. Electron tomography In single particle analysis, although you do not need crystals any more, you still average different particles, assuming they share the same structure. If not, the structural

3 variability and flexibility are averaged out. Single particle analysis does not work for highly heterogeneous systems like whole cells or organelles, in which molecules are stacked vertically. Electron tomography is used for such specimens. You record the micrographs of the same specimen from various view angles by tilting the stage of the microscope. Since you control the tilt angle, you have no angle-determination problem, although you still have to align the origin of images. However, the resolution is limited because of multi radiation. Example: Eukaryotic flagella, whole cell tomography

4 References Many examples of structural biology by EM can be seen in Alberts et al. (2002) Molecular biology of the cell, forth edition, Garland Science. p (techniques), p (molecular motor research combining various methodologies including EM). There are some (but, not many) descriptions in Branden, C. and Tooze, J. (1999) Introduction to structural biology, second edition Garland. A good review on single particle analysis and electron tomography Baumeister, W. and Steven, A.C. (2000) Macromolecular electron microscopy in the era of structural genomics Trends in Biochemical Sciences 25, Mathematical discussion on optics (including phase contrast, spherical aberration) Born, M. and Wolf, E. (1980) Principles of optics Pergamon Press Text books on single particle analysis Frank, J. (2006) Three-dimensional electron microscopy of macromolecular assemblies Oxford University Press.

5 Our target: Biological macromolecules (Protein, Nucleic Acids) Transmission Electron Microscope From the material of Virginia Tech. From Molecular Biology of the Cell Fourth Edition TEM and light microscopy share the similar optical system Differences 1. LM uses a photon as a probe, whereas EM uses an electron 2. LM uses glass lenses, whereas EM uses electromagnetic lenses 1

6 Inside of the electron microscope must be vacuum. The level of the vacuum varies, depending on the components (gun, column, camera etc.), and different pumps are used. Material Science You can see atoms directly Transmission Electron Microscopy (TEM) From the material of Virginia Tech. Biological Science 1. Low Contrast 2. High Sensitivity to the Electron Beam Why can t we see the atom directly in biological TEM? Wave particle duality of electrons Interference of waves Screen L de Brogli proposed and G. P. Thomson proved that electron has wave-particle duality. 2

7 Amplitude contrast (very weak with cryo-em) Phase contrast Defocus: interference generates contrast In focus: no interference, no phase constrast 3D reconstruction is equivalent to filling 3D Fourier space with many sections, determining the angles Various specimen preparations for biological macromolecules We would like to observe molecules as close to the physiological conditions as possible. Mica platinum Mica Carbon Carbon stain Shadowing Negative Staining Cryo (ice embedding) Baumeister, W. and Steven, A.C. (2000) 3

8 Cryo-EM: Ice-Embedding Cryo-holder Holey carbon grid Resolution 3 Å 8 Å Biological specimen (especially ice-embedded) is very sensitive to the radiation damage 6 e - /Å 2 30 e - /Å 2 15 Å 20 Å 6e - /Å 2 30 e - /Å 2 Conway et al. (1993) 4

9 2D crystal Gyobu et al., (2006) J. Struct. Biol. 146, 325. Stahlberg, H. et al. (2001) 3D view angles can be determined by diffraction pattern 2D crystal: electron microscopy and electron diffraction Missing wedge, missing pyramid and missing cone Gonen et al. (2005) Nature 438, 633. Molecular Biology of the Cell Lucic et al. (2005) Annu. Rev. Biochem. 5

10 Fourier Tubular crystal Kikkawa, T. et al. (1995) Diffraction of the tubular crystal is layer lines Acetylcholine receptor Unwin (1993) J. Mol. Biol. 229, 1101 Example of dynamic structural change: Actin / Myosin complex Tubular crystal has no missing information. Miyazawa et al. (2003) Nature 423, 949. Whittaker et al. (1995) Nature, 378,

11 Single particle analysis: 2D averaging Advantage to reconstruct without crystallization: Free solution condition Example of huge complexes: Ribosome Clp enzyme w/o substrate with substrate (before reaction) ATP hydrolysis Spahn et al. (2001) 107, 373. A P A with substrate (after reaction) Ishikawa et al. (2001) 20 nm Single Particle Analysis: Projection Matching Substrates ClpA 3D Reconstruction Initial Model Project Reproject Iteration ClpP Classification based on Cross Correlation Subaverage for each Euler angle 3D Reconstruction (New Reference) Data Final Reconstruction 5 nm 7

12 Electron Tomography of Chlamydomonas Flagella Gold clusters as marker for translational alignment -60 deg. 30 deg. 0 deg. 60 deg. In electron tomography, you have already known the view angle of images. But, images must be aligned translationally. 8

13 3D alignment and averaging improve S/N 3D Reconstruction of Chlamydomonas Flagella by electron cryo-tomography 3D Reconstruction of Chlamydomonas Flagella by electron cryo-tomography Averaging with 96nm periodicity reveals the IDA/ODA/RS/MT structure 96nm Outer dynein arm (accelerator, force generator) Radial spoke Inner dynein arm (regulator) Microtubule Bui et al. (2008) J. Cell Biol. 183, 923 9

14 Tail and AAA-ring arrangement of inner and outer arm dyneins Missing wedge, missing pyramid and missing cone Proxymal(-) Distal(+) Tails extend from the rings toward the distal end (tip of the flagellum, + end) Bui et al. (2008) J. Cell Biol. 183, 923 Baumeister, W. and Steven, A.C. (2000) Example: Whole cell tomography Missing wedge problem can be avoided by averaging In the particular case of flagella, structural information will be recovered for the whole Fourier space by averaging nine microtubule doublets. 10

15 Combination of high-resolution single particle analysis and medium-resolution electron tomography: Herpes simplex virus Single particle analysis with multirefrence for 8.5A reconstruction of procapsid by single particle analysis Zhou et al. (2000) Science 288, 877. coexisting structures: maturation of herpes simplex virus Heymann et al. (2000) Nat. Struct. Biol. 10, 334. More intact structure Lower dose Higher resolution tomography single particle analysis tubular crystal 2D crystal Electron tomography to see the whole virus (capsid, tegument, membrane) Resolution of various methodology of 3D cryo-tem Various Methods to reconstruct 3D structure of biological Molecules by EM Grunewald et al. (2003) Science 302,