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.

1. Introduction 372

1.1 Residual dipolar couplings as a route to structure and dynamics 372

1.2 A brief history of oriented phase high resolution NMR 374

2. Theoretical treatment of dipolar interactions 376

2.1 Anisotropic interactions as probes of macromolecular structure and dynamics 376

2.1.1 The dipolar interaction 376

2.1.2 Averaging in the solution state 377

2.2 Ordering of a rigid body 377

2.2.1 The Saupe order tensor 378

2.2.2 Orientational probability distribution function 380

2.2.3 The generalized degree of order 380

2.3 Molecular structure and internal dynamics 381

3. Inducing molecular order in high resolution NMR 383

3.1 Tensorial interactions between the magnetic field and anisotropic magnetic susceptibilities 383

3.2 Dilute liquid crystal media: a tunable source of order 384

3.2.1 Bicelles : from membrane mimics to aligning media 385

3.2.2 Filamentous phage 387

3.2.3 Transfer of alignment from ordered media to macromolecules 388

3.3 Magnetic field alignment 389

3.3.1 Paramagnetic assisted alignment 389

3.3.2 Advantages of using magnetic alignment 389

4. The measurement of residual dipolar couplings 391

4.1 Introduction 391

4.2 Frequency based methods 392

4.2.1 Coupling enhanced pulse schemes 392

4.2.2 In phase anti-phase methods (IPAP): 1DNH couplings in proteins 393

4.2.3 Exclusive correlated spectroscopy (E-COSY): 1DNH, 1DNC′ and 2DHNC′ 395

4.2.4 Extraction of splitting values from the frequency domain 396

4.3 Intensity based experiments 397

4.3.1 J-Modulated experiments: the measurement of 1DCαHα in proteins 397

4.3.2 Phase modulated methods 399

4.3.3 Constant time COSY – the measurement of DHH couplings 399

4.3.4 Systematic errors in intensity based experiments 400

5. Interpretation of residual dipolar coupling data 401

5.1 Structure determination protocols utilizing orientational constraints 401

5.1.1 The simulated annealing approach 401

5.1.2 Order matrix analysis of dipolar couplings 402

5.1.3 A discussion of the two approaches 402

5.2 Reducing orientational degeneracy 403

5.2.1 Multiple alignment media in the simulated annealing approach 404

5.2.2 Multiple alignment media in the order matrix approach 405

5.3 Simplifying effects arising due to molecular symmetry 406

5.4 Database approaches for determining protein structure 407

6. Applications to the characterization of macromolecular systems 408

6.1 Protein structure refinement 408

6.2 Protein domain orientation 409

6.3 Oligosaccharides 413

6.4 Biomolecular complexes 415

6.5 Exchanging systems 416

7. Acknowledgements 418

8. References 419

Within its relatively short history, nuclear magnetic resonance (NMR) spectroscopy has managed to play an important role in the characterization of biomolecular structure. However, the methods on which most of this characterization has been based, Nuclear Overhauser Effect (NOE) measurements for short-range distance constraints and scalar couplings measurements for torsional constraints, have limitations (Wüthrich, 1986). For extended structures, such as DNA helices, for example, propagation of errors in the short distance constraints derived from NOEs leaves the relative orientation of remote parts of the structures poorly defined. Also, the low density of observable protons in contact regions of molecules held together by factors other than hydrophobic packing, leads to poorly defined structures. This is especially true in carbohydrate containing complexes where hydrogen bonds often mediate contacts, and in multi-domain proteins where the area involved in domain–domain contact can also be small. Moreover, most NMR based structural applications are concerned with the characterization of a single, rigid conformer for the final structure. This can leave out important mechanistic information that depends on dynamic aspects and, when motion is present, this can lead to incorrect structural representations. This review focuses on one approach to alleviating some of the existing limitations in NMR based structure determination: the use of constraints derived from the measurement of residual dipolar couplings (D).