1. Reaching the Information Limit in Cryo- EM of Biological Macromolecules: Experimental Aspects -Robert M. Glaeser and Richard J. Hall (2011)

2. 1. Light microscope r = 172 nm 2. Electron Microscope r =.003 nm theoretical r =.27 nm point to point in JEOL 2100 scope 3. Theoretical vs experimental limit of cryo-EM 4. Why is there a gap between them? 5. How to minimize the gap. (Mostly, ZPC) Resolving Power

3. How large the density difference must be for the signal to be ≥ 3σ? Density values are expressed as multiples of the density of water

4. Atomic resolution(3Å) information by Henderson (1995) Theoretically ideal condition : 1. Homogenous (identical) objects 2. perfect contrast transfer 2. noise free detector Specimen size to align properly : 40 kDa 1. Depends on the contrast (signal) & exposure Particle numbers for atomic res. : 12,000 1. e- exposure required for the image 2. e- exposure that damages the molecule

5. Atomic resolution structures for icosaheral virus Nikolaus Grigorieff and Stephen C Harrison (2011) Current Opinion in Structural Biology

6. Atomic resolution structures with low symmetry 1.GroEL (~840 kDa, prototypical group I chaperonin) by Ludtke et al. 2008 LHe, JEOL JEM3000SFF (300kV) ~ 4 Å from 20,401 particles with D7 (x14) and C7 (x7) symmetry using EMAN 2.Mm-cpn (~960 kDa, an archaeal group II chaperonin) by Zhang et al. 2010 LN2, JEOL JEM3200FSC (300kV) 4.3 Å from 29,926 with D8 (x16) symmetry using EMAN

7. 4.0-Å resolution cryo-EM structure of the mammalian chaperonin TRiC/CCT JEM3200FSC, 300kV, 101K, 1 Mda,101,000 particles, EMAN, 2fold (4.7Å with asym) Cong et al. (2010) PNAS

8. In practice : 1. Specimen size : o about 800 kDa 2. Particle numbers : o millions of asymmetric units 3. Why the large gap? o Radiation sensitive object (low SNR) o Imperfect contrast transfer o Beam-induced movements o Detector o Aligning particles (reconstruction)

9. Object Image Model Microscopy Reconstruction Biological specimen, Heterogeneity, & Thick ice Imperfect detector, Poor CTF & Sample movement CTF-correction, Classification & Alignment

10. Electron exposure 1.‘‘Everything under the electron beam would burn to a cinder.’’ - Gabor (1928) 2. Radiation sensitive biological specimen limits at 2,000 e-/nm2 (20 e-/Å2) for 300 keV 3.Low SNR 4.Currently available detectors enhance noise of the low-dose images due to the imperfect detective quantum efficiency

11. Ideal contrast transfer

12. The “Object” : Biological specimen in frozen- hydrated condition 1.Weak-phase model Thin specimen with light atoms Modifies only the phase of the transmitted wave and not its intensity 2.Modest phase shift & low amplitude contrast  Low SNR 3.To enhance the contrast (signal)  Longer exposure  Averaging  Defocus  Phase Plate

13. Defocus (under focus) 1.Enhance the contrast 2.Poor signal transfer 1 um√3 um coherent incoherent Frank (2006)

14. A quarter-wave plate to apply a 90 phase shift to the scattered wave relative to the unscattered wave (Zernike, 1955) * Phase contrast is stronger at in-focus than defocus. Taylor series and assuming Φ(r) << 1, Phase shift by an object C Phase-shifted wave function 2007, Frank

15. Phase plate (Zernike phase contrast cryo-electron microscopy)

16. Chang et al. (2010) Structure : Simulated pol II images In-Focus Enhanced contrast  Higher SNR  Especially at low resolution

17. Simulated images of a 100 kDa enzyme embedded in vitreous ice

18. Epsilon15 Bacteriophage by Murata et al. (2010)

19. 56001500500100

20. Minimizing beam-induced movement

21. Bacteriorhodopsin 2D crystal

22. Beam-induced movement : 70S ribosomes

23. Efforts to minimize the movement 1. Limiting the size of illuminated area. 2. Improving the electrical conductivity of the support film. 3. However, only partial reduction has been achieved.

24. Calculated Fourier transform of the image of a monolayer crystal of paraffin grown on a 35-nm-thick carbon film Three sets of quasi-hexagonal reflections, all at a resolution of ~0.4 nm, have essentially the full, theoretically expected amplitude THE GOAL

25. l Barriers for the information limit and how to reach it l 1. Imperfect DQE of the detector  Noise-free detector l Pixilated electron counter l 2. Poor CTF  Ideal phase-contrast transfer function l Charging-free quarter-wave plate l 3. Beam-induced movement  ??!! l 4. Reconstruction CTF-correction Reliable classfication (different conformational states) l Perfect alignment