Skip to main content Accesibility Help
×
×
Home

Single-particle electron cryo-microscopy: towards atomic resolution

  • Marin van Heel (a1), Brent Gowen (a1), Rishi Matadeen (a1), Elena V. Orlova (a1), Robert Finn (a1), Tillmann Pape (a1), Dana Cohen (a1), Holger Stark (a1) (a2), Ralf Schmidt (a3), Michael Schatz (a1) (a3) and Ardan Patwardhan (a1)...
    • Published online: 01 March 2001
Abstract

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.

  • View HTML
    • Send article to Kindle

      To send this article to your Kindle, first ensure no-reply@cambridge.org is added to your Approved Personal Document E-mail List under your Personal Document Settings on the Manage Your Content and Devices page of your Amazon account. Then enter the ‘name’ part of your Kindle email address below. Find out more about sending to your Kindle. Find out more about sending to your Kindle.

      Note you can select to send to either the @free.kindle.com or @kindle.com variations. ‘@free.kindle.com’ emails are free but can only be sent to your device when it is connected to wi-fi. ‘@kindle.com’ emails can be delivered even when you are not connected to wi-fi, but note that service fees apply.

      Find out more about the Kindle Personal Document Service.

      Single-particle electron cryo-microscopy: towards atomic resolution
      Available formats
      ×
      Send article to Dropbox

      To send this article to your Dropbox account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Dropbox.

      Single-particle electron cryo-microscopy: towards atomic resolution
      Available formats
      ×
      Send article to Google Drive

      To send this article to your Google Drive account, please select one or more formats and confirm that you agree to abide by our usage policies. If this is the first time you use this feature, you will be asked to authorise Cambridge Core to connect with your <service> account. Find out more about sending content to Google Drive.

      Single-particle electron cryo-microscopy: towards atomic resolution
      Available formats
      ×
Copyright
Corresponding author
Author to whom correspondence should be addressed.
Recommend this journal

Email your librarian or administrator to recommend adding this journal to your organisation's collection.

Quarterly Reviews of Biophysics
  • ISSN: 0033-5835
  • EISSN: 1469-8994
  • URL: /core/journals/quarterly-reviews-of-biophysics
Please enter your name
Please enter a valid email address
Who would you like to send this to? *
×

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed