CIMST Winter School 2010 Lecture
Electron cryo-tomography of biological macromolecules
21 January 2010, 9:00-9:45 Dr. Takashi Ishikawa HPK F18 Tel: 633 2462 e-mail: email@example.com Lab webpage: http://www.mol.biol.ethz.ch/groups/ishikawa_group 1. Introduction: What can you see by electron microscopy ? For biological studies, transmission electron microscopy (TEM) is suitable for objects with dimension from 2 Å to 10 μm (fig.1). Electron microscopy for material science, in which you can visualize even one single atom, and that for biology use the same instrument, in principle. However, there are a number of differences in operation of the microscope, specimen preparation, analysis and interpretation of the images. These differences come from the low contrast (the density of protein ~ 1.35 against that of water ~ 1.0) and the high sensitivity. Even in the area of biological TEM, there are various different techniques; for cell biology and for molecular biology specimen preparations are quite different (fig.2). In this lecture, we discuss electron tomography and other methodologies of TEM for molecular biology. Electron microscopy is, unlikely to crystallography or NMR, direct imaging. Instead of diffraction or a spectrum, images of biological macromolecules are recorded. Since you do not need crystals, you can choose solution conditions freely, which enables you to visualize the molecular structure in dynamic motion close to or at in vivo conditions as well as molecular structure during dynamic motion. 2. Practical aspects 2-1. Specimen preparation Since the inside of the electron microscope is under vacuum, the specimen must be stabilized (fixation), to prevent it from being completely dried and destroyed. There are two classical approaches, in which solid templates of sensitive biological molecules are made: shadowing and negative staining (fig.3). Although high contrast can be obtained and the specimen is robust against electron beam, since templates are observed, you can see only the surfaces of molecules with these methods. To extend our view to the whole structure of molecules (including inner structure) in intact conditions, the technique of cryo-EM was developed, in which molecules are quickly frozen by liquid ethane (~ -170 degrees) and observed in the condition of the ice-embedded state (figs. 4,5). Freezing reduces radiation damages as well, which is regularly done in X-ray crystallography, too.
Which enable the high quality data from lower illumination (i. this results in loss of contrast.8).10 optional) Molecules are arrayed as spirals (fig. You must determine these two unknown factors at the same time.6). for example (fig. One approach is shown
. the resolution is not so high as 2D crystals. 3. determining their angles by computation. you do not know either the final 3D structure or the view angles of individual particles. later). you can only see subdomains (fig. Tubular crystal (figs. the resolution of biological EM is much more limited. Resolution and radiation damage The resolution describes how fine the information you have is. However. in the Fourier space. which allows you to see different stages of reaction. pre-processes like focusing must be done in the different place from the specimen (fig. you can earn higher signal-to-noise ratio. Example: acetylcholine receptor. the angle determination is complicated. Advantage. you can set solution condition freely. Initially. However. we must pay effort to minimize radiation damage (fig. is that we do not need heavy atoms. Thus.9) 3-3.e. At ~20 Å resolution.10). To minimize the radiation damage before the data collection. Unfortunately.9. Single particle analysis This is a technique to calculate the 3D structure from many single particles (>5. Good compromise must be found. Therefore. because of the poor contrast and sensitivity of the specimen. For 3D information. 2-2. You need ~2 Å to distinguish the direction of side chains. optional) In the 2D crystal molecules array two-dimensionally. View angles can be judged from the diffraction pattern. comparing to X-ray crystallography. resulting higher resolution (more than ten structures have been solved at the atomic resolution).9). as X-ray crystallography. Although in material science you can see atoms or even electron orbitals directly by EM.6). 8 Å resolution is enough for secondary structures (α-helices and β-sheets). Since the molecules are dispersed in the solution. we must minimize the dose (= the amount of electron radiation).For cryo-TEM. You need ~3 Å resolution to trace individual amino acids. Since the signal will be spread along one dimensional lines. 3-1. tilted 2D crystals are inserted into the microscope with special holder. crystallization. 3-2.11). The Fourier transform of the image is a lattice. Since you cannot tilt 2D crystal at 90 degrees in the microscope. 2D crystal (fig. All the necessary view angles for 3D reconstruction can be obtained from tubular crystal. microtubule (fig. there is no missing information and tilt is not needed.000). actin (fig. this methodology demands. lower radiation damage).9.7). 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. because direct images are recorded. you will miss some information in the 3D Fourier space (missing cone. As biological specimens are easily broken under electron illumination (fig.
13). Limitation of tilt angles causes artifacts called missing wedge (figs. Example: Clp protease (fig.15-23).18-20).21-23). Example: Eukaryotic flagella (figs. you can average them to earn signal-to-noise ratio. Since you control the tilt angle.15). the structural variability and flexibility are averaged out (summarized in fig. If not. which starts from a certain template and find the bestfitted view angle for each raw data particle.14). whole cell tomography (fig. Single particle analysis does not work for highly heterogeneous systems like whole cells or organelles. you have no angle-determination problem.24)
. In the case you can detect molecules with identical structure. Electron tomography is used for such specimens. assuming they share the same structure.16). although you still have to align the origin of images (fig. which also compensate missing wedge (figs. Electron tomography In single particle analysis. You record the micrographs of the same specimen from various view angles by tilting the stage of the microscope (fig. However. you still average different particles. in which molecules are stacked vertically.14). although you do not need crystals any more. 3-4. the resolution is limited because of multi radiation. ribosome (fig.here: projection matching (fig.17).
. There are some (but. enhancement and interpretation” North-Holland. 537-543. J. (1999) “ Analysis of macromolecular structure and dynamics by electron cryo-microscopy” Curr. Opin.A. Garland Science. (2002) “Molecular biology of the cell. J.References Many examples of structural biology by EM can be seen in Alberts et al. K. forth edition”. p.C. Text books on single particle analysis Frank. 949-969 (molecular motor research combining various methodologies including EM). A. C. and Williams. Frank.7: Image analysis. Examples of high resolution electron microscopy with crystals Kuhlbrandt W. (1999) “Introduction to structural biology. Biol. 624-631. (2000) “Macromolecular electron microscopy in the era of structural genomics” Trends in Biochemical Sciences 25.L. (edited) (1992) “Electron tomography” Plenum Press. (1978) “Practical methods in electron microscopy. and Steven. not many) descriptions in Branden. Technical aspects Misell. vol. A good review on single particle analysis and electron tomography Baumeister. and Tooze. 3. D. W. (1996) “Three-dimensional electron microscopy of macromolecular assemblies” Academic Press. J. Chem. second edition” Garland. 560-570 (techniques).
From “Molecular Biology of the Cell” Fourth Edition
Transmission Electron Microscopy (TEM)
Kirreva et al.2
Kirreva et al. (2004) Bar = 1 μm
Fig. Nucleic Acids)
Fig. (2004) Bar = 1 μm
Schalch et al.Our target: Biological macromolecules (Protein.
Cryo (ice embedding)
Holey carbon grid
Fig.Various specimen preparations for biological macromolecules
We would like to observe molecules as close to the physiological conditions as possible.
Biological specimen (especially ice-embedded) is very sensitive to the radiation damage
Conway et al. (1993)
Low dose: To illuminate specimen only for data collection
949. et al. Crystallization 2D crystal
Stahlberg. (2003) Nature 423. (1995)
Kikkawa.Various Techniques for 3D Reconstruction
Miyazawa et al. (2001)
. et al. T.
Single particle analysis: 2D averaging Advantage to reconstruct without crystallization: Free solution condition
Clp enzyme w/o substrate
ATP hydrolysis with substrate (before reaction)
with substrate (after reaction) Ishikawa et al. 373.12
Fig. (2001) 107.
Example of huge complexes: Ribosome
Spahn et al.
Single particle analysis Principle to obtain multiple views Crystallization Structural heterogeneity Current resolution Missing information Merged many particles with various views in solution Not needed Averaged out High (up to 8 Å) None / Missing cone
Electron tomography Take micrographs of one particle tilted at various angle in the microscope Not needed Visualized individually Low (currently 50 Å) Missing wedge
.Single Particle Analysis: Projection Matching
Initial Model Project Reproject
Classification based on Cross Correlation Subaverage for each Euler angle 3D Reconstruction (New Reference)
Gold clusters as marker for translational alignment
30 deg. But.15
In electron tomography. images must be aligned translationally.16
. you have already known the view angle of images.
Baumeister. missing pyramid and missing cone
Fig. and Steven.17
Tomogram of flagellum in vertical and horizontal sections
Missing wedge.a b c
Fig.C. A. W. (2000)
. structural information will be recovered for the whole Fourier space by averaging nine microtubule doublets. (2005) Annu. missing pyramid and missing cone
Missing wedge problem can be avoided by averaging
Fig.Missing wedge. Biochem.19
Lucic et al. Rev.20 In the particular case of flagella.
(Bui et al.21
Flagella structure averaged from Cryo-Electron Tomogram
Wild Type 24nm
Outer dynein arm: Accelerator Microtubule: skeleton Inner dynein arm. Dynein regulatory complex: Regulator Radial spoke: transducer 96nm
ODA1 mutant (no outer dynein arm) ODA11 mutant (no α-chain of outer dynein arm)
Microtubule/dynein structure averaged from electron tomography
Fig. unpublished data)
368.Electron tomography from various mutants reveals the protein network in flagella
Docking complex: outer dynein arms are anchored
Connection between outer and inner dynein arms (Bui et al. (2007) J. Mol. 1249)
Stalk: connection between adjacent microtubules
Example: Whole cell tomography
Fig. unpublished data)
Tails of outer dynein arms: responsible for cooperativity
(Ishikawa et al.