Single-particle electron cryo-microscopy: towards atomic resolution
Authors: van Heel Marin , Gowen Brent , Matadeen Rishi , Orlova Elena V. , Finn Robert , Pape Tillmann , Cohen Dana , Stark Holger , Schmidt Ralf , Schatz Michael , Patwardhan Ardan 
Published: July 2002

1. Introduction 308

2. Electron microscopy 311

2.1 Specimen preparation 311

2.2 The electron microscope 311

2.3 Acceleration voltage, defocus, and the electron gun 312

2.4 Magnification and data collection 313

3. Digitisation and CTF correction 317

3.1 The patchwork densitometer 318

3.2 Particle selection 320

3.3 Position dependent CTF correction 321

3.4 Precision of CTF determination 321

4. Single particles and angular reconstitution 323

4.1 Preliminary filtering and centring of data 323

4.2 Alignments using correlation functions 324

4.3 Choice of first reference images 324

4.4 Multi-reference alignment of data 325

4.5 MSA eigenvector/eigenvalue data compression 328

4.6 MSA classification 330

4.7 Euler angle determination (‘angular reconstitution’) 332

4.8 Sinograms and sinogram correlation functions 332

4.9 Exploiting symmetry 335

4.10 Three-dimensional reconstruction 337

4.11 Euler angles using anchor sets 339

4.12 Iterative refinements 339

5. Computational hardware/software aspects 341

5.1 The (IMAGIC) image processing workstation 342

5.2 Operating systems and GUIs 342

5.3 Computational logistics 344

5.4 Shared memory machines 344

5.5 Farming on loosely coupled computers 346

5.6 Implementation using MPI protocol 347

5.7 Software is what it's all about 347

6. Interpretation of results 348

6.1 Assessing resolution: the Fourier Shell Correlation 348

6.2 Influence of filtering 351

6.3 Rendering 351

6.4 Searching for known sub-structures 352

6.5 Interpretation 353

7. Examples 353

7.1 Icosahedral symmetry: TBSV at 5·9 Å resolution 354

7.2 The D6 symmetrical worm hemoglobin at 13 Å resolution 356

7.3 Functional states of the 70S E. coli ribosome 357

7.4 The 50S E. coli ribosomal subunit at 7·5 Å resolution 359

8. Perspectives 361

9. Acknowledgements 364

10. References 364

In the past few years, electron microscopy (EM) has established itself as an important – still
upcoming – technique for studying the structures of large biological macromolecules. EM is
a very direct method of structure determination that complements the well-established
techniques of X-ray crystallography and NMR spectroscopy. Electron micrographs record
images of the object and not just their diffraction patterns and thus the classical ‘phase’
problem of X-ray crystallography does not exist in EM. Modern microscopes may reach
resolution levels better than ∼ 1·5 Å, which is more than sufficient to elucidate the
polypeptide backbone in proteins directly. X-ray structures at such resolution levels are
considered ‘excellent’. The fundamental problem in biological EM is not so much the
instrumental resolution of the microscopes, but rather the radiation sensitivity of the
biological material one wants to investigate. Information about the specimen is collected in the
photographic emulsion with the arrival of individual electrons that have (elastically)
interacted with the specimen. However, many electrons will damage the specimen by non-elastic interactions. By the time enough electrons have passed through the object to produce
a single good signal-to-noise (SNR) image, the biological sample will have been reduced to
ashes. In contrast, stable inorganic specimens in material science often show interpretable
details down to the highest possible instrumental resolution.