Cryoelectron Microscopy

1. Cryoelectron Microscopy Andy HowardBiology 5559 October 2018

2. Agenda • Electron Microscopy • Applying Transmission EM to biological macromolecules • Low temperature: why and how • Negative stains • Multiple-sample averaging

3. Electron Microscopy • Electrons are directed from a source toward a sample that absorbs and scatters them • Electro-optical lenses direct and focus the electrons • Transmitted electrons are detected with a film (ancient!) or an electronic detector • Hans Busch, Ernst Ruska developed first instruments • Overcomes diffraction limit of visual microscopy

4. Transmission electron microscopy • Tungsten tube sends e- toward sample, which scatters and absorbs them • Variations in transmission observed via electronic lenses and a phosphor that converts the image to light, which is detected, e.g. in a CCD • Sample must be thin enough not to absorb all of the beam or produce multiple scattering: < 100nm RCA desktop EM, 1950

5. Scanning electron microscopy • Focused beam raster-scanned across specimen • Sample converts electrons’ energy to heat, secondary e-, emission of EM radiation • Can produce 3-D reconstructed images even of thick objects, e.g. intact animals • 10x lower resolution than TEM

6. Scanning transmission electron microscopy • Like TEM in that it handles thin samples via absorption • But a focused, rastered beam is entrained on a sample • Can produce resolutions comparable to TEM Aecium with sporesCourtesy Charles Mims, Ball State U

7. Sample preparation • Various sample prep techniques increase contrast, hold sample in place, or enable examination under low temperatures or high pressures • Negative staining: specimen mixed with electron-opaque material like uranyl acetate, phosphotungstic acid, etc. • Cryogenics: see subsequent discussion

8. So what’s the problem? • If the sample is interesting, it has a propensity for absorbing as well as scattering electrons • That means it will be rapidly destroyed at room temperature • So we solve that by flash-cooling the samples • Much of the relevant technology is transferrable to cryo-crystallography

9. What else is a problem? • Room temperature biological samples don’t respond well to being introduced to the moderately high vacuum of a TEM grid • … they’re soft and fall apart if they’re exposed to vacuum • It turns out that operating at 100K protects the samples from this kind of damage too!

10. But wait: if you freeze the sample, won’t it get destroyed? • … not if you cryo-protect it and cool it fast! • Then the water surrounding the sample turns to vitreous ice rather than crystalline ice • Typically the sample is prepared in liquid ethane and then deposited on the TEM grid

11. So how good an image can you get? • From a single specimen: 1-10nm (10-100Å) resolution • That’s good enough to see a small virus as a blob, but won’t let us identify chains or individual domains of proteins or viruses • How can you do better? AVERAGING!

12. What do you mean by that? • Often we can deposit a 2-D array of specimens onto grid • They don’t have to be perfectly aligned relative to one another (unlike crystals!) • Once you obtain your image, you can average the various samples with respect to one another in software to obtain an with more detail than the individual images F-actin: Kim et al, JAST 1:159 (2010)

13. How much can that help? • Ideal conditions (e.g. with icosahedral viruses) allow for ~ 0.35nm (3.5Å) resolution • Compares favorably with early crystallographic studies of proteins • Can show helices if edge-on, for example • Definitely can show boundaries between subunits or domains

14. Applications • Distinguishing states of molecules as they are altered by binding of ligands or other macromolecules • Identifying components of molecular machines • Combinations with high-resolution techniques: see next section

15. Figure 1. Binding to sCD4 or 17b is sufficient for formation of the open quaternary Env conformation. HIV envelope as modified by binding of other proteins(Tran et al, PLOS Pathogens 8(7): e1002797) Tran EEH, Borgnia MJ, Kuybeda O, Schauder DM, et al. (2012) Structural Mechanism of Trimeric HIV-1 Envelope Glycoprotein Activation. PLoS Pathog 8(7): e1002797. doi:10.1371/journal.ppat.1002797 http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002797

16. CryoEM as an adjunct to other techniques • Solving the crystallographic phase problem by boot-strapping: • Use CryoEM to get a 6-4Å structure of a protein or virus • Collect 2.5-1.5Å crystallographic data • Calculate phases based on 6-4Å EM model • Gradually extend phases to 2.5-1.5Å by careful model-building

17. Example: bacteriophage PM2:Stuart et al. (2013), Acta Crystallographica D69:2257

18. Cryo-EM plus component crystallography • Often a multi-polypeptide complex can be imaged at intermediate resolution via cryoEM • Individual components of complex can be crystallized and structures of those components determined by crystallography • … or individual components could have their structures determined by NMR • Then you fit those higher-resolution objects into the intermediate-resolution EM model

19. How subtle can that be? • Clearly the components may undergo conformational changes when they form the multi-subunit complex • Often these changes can be modeled within the EM image if it’s at high enough resolution • … or other experimental techniques can provide clues as to how the interaction changes the components • … or we use molecular dynamics to model those changes!

20. Recent innovations • Huge improvements in detector quality • Better sample prep techniques • Significant software improvements make data management and structure determination feasible for non-specialists • See Nobel talk (starting now!) for specifics