1. Growth of EM Method for Determining Structures of Macromolecular Assemblies

2. What is cryo EM? EM = (Transmission) Electron Microscopy
Cryo EM = technique where biological samples are preserved in vitreous ice and imaged by EM at cryogenic temperatures.
EM reconstruction = 3D maps are generated by averaging over many EM images.

3. From Sample to Structure
Slide from Wah Chiu, Baylor

4. Reconstruction Method
Sample Types
Type
Examples
Reconstruction Method
Typical
resolution
Single particles
Icosahedral viruses, GroEL, ribosome
Single particle reconstruction
20-4 Å
Filaments
Flagella, filamentous viruses, actin, tubular crystals
Helical reconstruction
15-3 Å
2D crystals
Catalase, aquaporin, tubulin
2D electron crystallography
10-2 Å
Ensembles
HIV capsids
Electron Tomography
40-20 Å

5. Single Particles MW lower limit: ~200 kDa Icosahedral virus Ribosome
Image sources: EMDB, Joachim Frank, Sacha De Carlo

6. Filaments Acetylcholine receptor tubular crystal Tobacco Mosaic Virus
gingi.uchicago.edu/achr.html
Actin-myosin complex
ami.scripps.edu

7. 2D Crystals Aquaporin 2D crystals, electron diffraction
Lipid-protein interactions
Gonen et al Nature 438:633-8.

8. Ensembles Reconstruction of HIV capsids by cryoEM tomography
Benjamin et al J. Mol Biol. 346:

9. specimen preparation SUPPORT GRID 3.05 mm 400 divisons/inch
1 division/66 microns
Copper or other metal
Manufactured “holey carbon grid”
Holes typically 2 microns diameter
2spi.com/catalog

10. Negative Stain vs. Vitreous Ice
Specimen in Stain
Cryogenic Specimen
vitreous ice layer
uranyl acetate
High contrast image
No special temperature control
Essentially no radiation damage
Particle distorted
Image = stain “shell” around the particle
Low resolution method: Å
Great choice for initial sample screening
Low contrast image
Sample maintained at cryogenic temperature (85 K)
High radiation damage
Particle undistorted
Image is of the actual particle
Higher resolution obtained: 15-4 Å
Best choice for reconstruction

11. Imaging--TEM microscope
Sample
Goes here
Swap out
to view diffraction
Swap out screen
to record image
Film/CCD camera
cryoem.berkeley.edu/~nieder

12. Cryo EM Experiment Characteristics
Images must be taken with low electron doses to prevent radiation damage
Averaging over thousands of individual particles and/or “asymmetric units”
Image:

13. Data Collection and Initial Image Processing
Collect image set ( images, vary focus)
Pick Particles ( ,000)
Perform contrast-transfer-function (CTF) correction for each image
Center, align, classify, make “class averages”

14. Particle Selection Raw image Auto-Select particles Particle Composite
For 1 raw image

15. CTF Correction

16. Class Averages

17. Recovering 3D from 2D
figure from Joachim Frank

18. Reconstruction Cycle
cryoem.berkeley.edu/~nieder
Final map

19. Map Quality
figure from Joachim Frank

20. Methods to interpret cryo EM map volumes:
Structure Analysis
Methods to interpret cryo EM map volumes:
“segmentation” -- identifying different parts of the map
“fitting” --placing atomic coordinates into the map, e.g., from X-ray structures
very new: normal mode refinement to improve fit

21. Segmentation herpes simplex capsid heterotrimer
Epsilon 15 bacteriophage
Images from Wah Chiu

22. Fitting Rossmann, M. G. et al 2004. Curr Opin Struct Biol 14:171-80.
Kostyuchenko, V. A. et al 2005 Nat Struct Mol Biol 12:810-3.

23. Soon…a single archive site for maps and models
Archiving
wwpdb.org
Coordinates
Maps
Soon…a single archive site for maps and models

24. References The Definitive Textbook:
Frank, J Three-dimensional electron microscopy of macromolecular assemblies : visualization of biological molecules in their native state. 2nd ed. New York : Oxford University Press.
Reviews:
Chiu, W., M. L. Baker, and S. C. Almo Structural biology of cellular machines. Trends Cell Biol 16:
Nickell, S., C. Kofler, A. P. Leis, and W. Baumeister A visual approach to proteomics. Nat Rev Mol Cell Biol 7:

25. Resources
Research-Resource Centers for Molecular Microscopy:
EM software tools: en.wikipedia.org/wiki/Software_tools_for_molecular_microscopy
Visualization:
EMDB map database:
Icosahedral Viruses: viperdb.scripps.edu

26. Cool Movies based on cryoEM data
Microtubules:
T4 virus:
Ribosome:
Actomyosin/kinesin:
T4 virus