Specimen Preparation and Imaging for Macromolecular Electron Microscopy Gina Sosinsky Neu 259

1. Specimen Preparationand Imaging forMacromolecular Electron Microscopy Gina Sosinsky Neu 259 May 29, 2012

2. Topics • Range of sample sizes studied by macromolecular microscopy • Types of samples (Repeating assemblies) • Never-ending quest for higher resolution • What limits resolution? • Techniques • Metal shadowing • Negative/positive staining • Embedding in sugars or tannic acid • Cryo-Electron microscopy • Cryo-Negative staining • Cryo-tomography • Low dose microscopy

3. Range of Sample Sizes Studiedby Macromolecular Microscopy Paramecium bursariaChlorella Virus 1~1900 Å, ~1 GDa Caulobacter crescentus~0.6 X 2 µm Bacteriorhodopsin~58 Å, ~26 kDa 70S E. coli ribosome~250 Å Theoretical Biophysics Group Beckman Institute University of Illinois at Urbana-Champaign Timothy S. Baker Group UCSD Briegel, et al. (2006) Courtesy J. Frank 100 Å 1000 Å 1 µm

4. Types of Samples Studiedby Macromolecular Microscopy Repeating Assemblies 70S E. coliribosome Hepatitis B virus core Actin-myosin filament Light-harvesting 2D crystal Single particles with little or no symmetry Single particles with icosahedral or other symmetries Helical symmetry Two-dimensional crystals Baker and Henderson (2001)

5. Types of Samples Studiedby Macromolecular Microscopy Cells, Organelles, Pleomorphic Viruses, etc. Human Immunodeficiency Virus - 1Zhu, et al. (2006) Baker and Henderson (2001) Nuclear Pore ComplexBeck, et al. (2007)

6. The Never-Ending Quest for Higher Resolution in EM Points of Resolution in Structural Information of Proteins Fujiyoshi (1998) Adv. Biophys 35:25-80

7. Limits of Resolution of Various Imaging Technologies The Never-Ending Quest for Higher Resolution in EM & NMR Currently achieved resolution Leis, et al. (2009) Prediction for resolution improvement

8. The Never-Ending Quest for Higher Resolution in EM Resolution of Selected Solved Structures

9. The Never-Ending Quest for Higher Resolution in EM Visualizing Helices in Penicillium stoloniferum Virus ~ 350 Å Diameter~7.3 Å resolution X-eyed Stereo

10. What Limits Resolution? • The vacuum of the microscope - DEHYDRATION! • ~9 X 10-8 Torr or 1 X 10-10 atm • Look at the lengths we go to avoid this! • We dehydrate it with solvents • We flash freeze it and then freeze dry it • We embed it in heavy metals • We vitrify it (Flash frozen in amorphous ice)

11. What Limits Resolution? • The vacuum of the microscope - DEHYDRATION! • Lack of contrast - BIOLOGICAL SAMPLES JUST DON’T DO A VERY GOOD JOB AT SCATTERING ELECTRONS!

12. What Limits Resolution? • The vacuum of the microscope - Dehydration • Lack of contrast - Biological samples just don’t do a very good job at scattering electrons • Radiation damage - Biological samples just don’t like getting hit by the electron beam

13. Radiation Damage - Primary and Secondary Effects • Primary Effects • Temperature independent • Occurs in the first 10-14 seconds • Excitation of orbital electrons of the sample forms ions and radicals • Secondary Effects • Secondary effects are what cause damage in the sample • Secondary effects are temperature dependent • Chemical and physical changes (breaking C-H and C-C bonds) • Mass loss (can be as great as 50%) • Charging effects • Contamination • Residual hydrocarbons in the vacuum chamber can break into fragments that become deposited on specimens. • Water vapor from insertion of the sample and from photographic film • Loss of order in crystalline specimens

14. Radiation Damage - Loss of Order in Crystalline Specimens Changes in the electron diffraction pattern of frozen-hydrated catalase crystals resulting from radiation damage <1 e-/Å2 2.5 e-/Å2 2.8 Å 5.0 e-/Å2 11 e-/Å2 Example Dosage: The minimum dosage necessary to see an image on the screen at 20kX magnification is ~4e-/Å2/sec. 8.5 Å Taylor and Glaeser (1976) J. Ultrastruc. Res. 55:448

15. 10 e-/Å2 20 e-/Å2 30 e-/Å2 40 e-/Å2 Radiation DamageFrozen-hydrated Simian Virus 40 Dose Series

16. Techniques • Metal shadowing • Negative/positive staining • Embedding in sugars and tannic acid • Cryo-electron microscopy • Cryo-negative staining • Cryo-tomography

17. Techniques - Metal Shadowing Wischnitzer (1970) Introduction to electron microscopy, 2nd ed.

18. Techniques - Metal Shadowing • Problems • Only see the surface • Standard techniques are low resolution, evaporated metals tend to be granular • Metal decoration Actin + S1, Courtesy of John Heuser

19. Techniques - Negative/Positive Staining • “Negative stain” is a misnomer. Most of the stain fills in sample depressions thereby preventing sample collapse. It does not stain the sample. • Sample appears “white” and the electron-dense stain is “black”. • Helps to reduce dehydration and radiation damage effects. • Attainable resolution is ~ 15-25 Å (~10 Å - GroEL De Carlo et al. 2008). 8 Å for crystals. • Positive staining occurs when ions of the stain react with the molecule. Hayat & Miller (1990)Negative Staining

20. Techniques - Negative/Positive Staining Examples of Commonly Used Negative Stains • Others: • Methylamine tungstate • Silver nitrate • Aurothioglucose • Sodium tetraborate • Cadmium iodide

21. Techniques - Negative/Positive Staining Choosing the Proper Stain • High density • 3.8 - 5.7 gm/cc versus 1.37 gm/cc for protein • High solubility • High melting and boiling points • Stability in the beam • Stain needs to have a fine granularity (0.4 to 1.5 nm) • No chemical reaction with the specimen • Choice of stain may depend on the pH and salt concentration needs of the sample

22. Carbon-filmed grid Techniques - Negative/Positive Staining Procedure - Setup dH2O Sample Filter paperwedges 1% UA stain in water Grid Box

23. Techniques - Negative/Positive Staining Procedure - Hydrophilic Carbon Surface Hydrophobic surface Hydrophilic surface

24. Techniques - Negative/Positive Staining Procedure - Glow Discharging the Grids

25. Techniques - Negative/Positive Staining Procedure - Apply Sample to Grid

26. Techniques - Negative/Positive Staining Procedure - Wash with dH2O

27. Techniques - Negative/Positive Staining Procedure - Apply Stain

28. Techniques - Negative/Positive Staining Procedure - Blot with Filter Paper

29. Techniques - Negative/Positive Staining Results Bacteriophage T4 Maize Streak Virus

30. Techniques - Negative/Positive Staining Problems • Unpredictable and uneven staining • High contrast images only the surface • Sample flattening • The electron beam can redistribute the stain • Different stains give different views Parmecium bursariaChlorella virus Uranyl acetate stained Cryo-electron microscopy

31. Techniques - Negative/Positive Staining Artifacts “The question of what is artifact and what is not is a persistent one in electron microscopy, especially when micrographs depict what are essentially newer unexplored structures.“ Dr. Keith Porter, 1979. From Heuser (2002)

32. Techniques - Embedding inSugars or Tannic acid • Making the preparation is similar to that of negative staining. • Specimen is supported by a matrix of concentrated sugar to maintain the need for hydration. • Often used with crystalline samples in order to keep them flat on the grid • Very beam sensitive, often need to keep sample at liquid nitrogen temperature • Very low contrast, sugar scatters electrons as well as protein (contrast matching)

33. Techniques - Cryo-Electron Microscopy (CryoEM) • Specimens are frozen in non-crystalline (vitreous ice). Freezing must be done in less than 10-4 sec. • Specimens are “frozen-hydrated”. This overcomes the problem of putting a hydrated sample in a vacuum. • Specimens are observed with “native contrast” - no staining, no fixatives. • Samples must be maintained below ~-140o C in the microscope. (below the vitreous to crystalline ice phase transition) • Maintains the native structure of the molecule to atomic resolution. Bacteriophage  29

34. Techniques - CryoEM Holey Carbon Support Film

35. Techniques - CryoEM Sample Preparation Equipment

36. Techniques - CryoEM Sample Preparation Equipment Guillotine Plunger EM tweezers LN2 dewar Foot switch

37. Techniques - CryoEM Sample Preparation Equipment Guillotine Plunger ethane EM forceps EM grid LN2 LN2 dewar

38. Techniques - CryoEM Addition of Sample and Blotting

39. Techniques - CryoEM Addition of Sample and Blotting Filter paper Filter paper EM grid Sample

40. LN2 (-196 °C) EM grid ethane Techniques - CryoEM Plunging the Grid into Ethane and Transfer to Nitrogen

41. Techniques - CryoEM Transferring to the Grid Storage Box

42. Techniques - CryoEM Automated Freezing with the FEI Vitrobot Environmental chamber Computer-controlled

43. Techniques - CryoEM Using the Vitrobot

44. EM tweezers Sampleon grid Filter paper disks Techniques - CryoEM Using the Vitrobot

45. Techniques - CryoEM Using the Vitrobot

46. Techniques - CryoEM Using the Vitrobot

47. Techniques - CryoEM Cryo-transfer Workstation and Holder Cover Transfer Area Cryo-Shield Nitrogen Dewar Workstation

48. Techniques - CryoEM Transferring the Grid into the Cryo-holder Grid Box Cryo-Shield Grid Clipring

49. Techniques - CryoEM Transferring the Grid into the Cryo-holder Grid Box Cryo-Shield Grid Clipring

50. Techniques - CryoEM Transfer of the Grid to the Cryo-holder