Cryo-electron microscopy Cryo-EM. Garry Taylor
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1 Cryo-electron microscopy Cryo-EM Garry Taylor
2 Electron has a wavelength de Broglie relationship: m v = h / λ or λ = h / mv Accelerate e - in a field of potential V, it gains energy ev which is converted to kinetic energy: 1/2 mv 2 = ev or v = ( 2eV/m) 1/2 λ = h / (2emV) 1/2 V (kv) λ(å)
3 Scanning electron microscopy SEM ~100Å beam swept across the surface ~2000Å resolution
4 Transmission Electron Microscopy (TEM) 100Kev to 1 Mev electrons illuminate whole field of view. Image is a projection of the thin specimen.
5 TEM electrons generated & focused specimen objective lens (forms image) intermediate lens (switch between imaging and diffraction modes) projector lens (magnifies image or diffraction pattern) image
6 TEM
7 Interactions of electrons with matter 90% pass through Elastic scattering Electron interacts with Coulomb potential of nucleus (2000 x heavier) Bounces off, no energy loss, same λ Inelastic scattering Electrons interact with electrons Energy loss, different λ, focused at different place Chromatic aberration Radiation damage - ions & reactive species So, use very thin specimens
8 Negative stain v cryo-em
9 Vitrification - rapid freezing
10 Three main methods of image reconstruction 1. Electron crystallography - images and electron diffraction patterns 2. Single particle analysis - identical particles viewed in different orientations 3. Electron tomography - multiple images of the same specimen recorded at different tilt angles
11 1. Electron crystallography need a regular 2D array of molecules image enhancement by Fourier averaging 2D object with local Typical noisy image Electron diffraction of 3-fold symmetry Fourier transform to noisy image - measure get phases intensities of peaks Combine diffraction amplitudes with image phases 3-fold symmetry average image
12 Electron crystallography from 2D to 3D 1. Tilt sample 2. Capture image 3. Collect electron diffraction 4. Build up 3D Fourier space (amplitudes from electron diffraction, phases from Fourier inversion of image) 5. Calculate 3D electron density map
13 First success..bacteriorhodopsin ~3.5Å resolution in the plane, lower resolution perpendicular to the plane
14 Extending the method.. inducing 2D arrays
15 Human aquaporin 4Å electron diffraction of ice-embedded 2D crystals Stereo view of six tilted transmembrane α-helices
16 ATPase at 8Å resolution
17 Tubulin at 3.7Å resolution
18 Tubulin
19 Three main methods of image reconstruction 1. Electron crystallography - images and electron diffraction patterns 2. Single particle analysis - identical particles viewed in different orientations 3. Electron tomography - multiple images of the same specimen recorded at different tilt angles
20 2. Single particle analysis
21 2. Single particle analysis Lower limit is kda. Missing cone not a problem as long as multiple views are present Actually 1000s of particles Problem: finding relative orientation of each particle First achieved with icosahedral viruses fold symmetry reduces number of particles needed. 9Å resolution at best.
22 Hepatitis B virus capsid 9Å icosahedral 3D reconstruction from single particles Model of the 149- residue dimer that serves as its building block
23 Poliovirus and its receptor 21Å resolution virus with water soluble ectodomain of its receptor revealed binding interactions allowed construction of quasi-atomic model of receptor, known to consist of 3 immunoglobulin domains
24 Interactions of the elongation factor with the 70S E. coli ribosome 20Å map of ribosome (blue) and EF-G in red, from difference map 13Å map of ribosome with EF-G and trnas
25 18Å resolution structure of the 12- subunit complex RNA polymerase
26
27 Conformational changes studied by Cryo-EM chaperone.html
28 Three main methods of image reconstruction 1. Electron crystallography - images and electron diffraction patterns 2. Single particle analysis - identical particles viewed in different orientations 3. Electron tomography - multiple images of the same specimen recorded at different tilt angles
29
30 3. Electron tomography Multiple projections of a particle at different angles Problem: electron dose ~1000 e nm -2 for high resolution ~10,000 e nm -2 for medium resolution
31
32
33 Cryo-ET of viruses Herpes simplex virus Vaccinia virus HIV virus 100nm 2-D image reconstructed tomogram slice colored cutaway 3-D image
34 3D structure of eukaryotic flagella in a quiescent state revealed by cryo-electron tomography
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