1. Introduction 1

1.1 The ‘band gap’ in silicon 2

2. Principles of CCD detector operation 3

2.1 Direct detection 3

2.2 Electron energy conversion into light 4

2.3 Optical coupling: lens or fibre optics? 6

2.4 Readout speed and comparison with film 8

3. Practical considerations for electron microscopic applications 9

3.1 Sources of noise 9

3.1.1 Dark current noise 9

3.1.2 Readout noise 9

3.1.3 Spurious events due to X-rays or cosmic rays 10

3.2 Efficiency of detection 11

3.3 Spatial resolution and modulation transfer function 12

3.4 Interface to electron microscope 14

3.5 Electron diffraction applications 15

4. Prospects for high-resolution imaging with CCD detectors 18

5. Alternative technologies for electronic detection 23

5.1 Image plates 23

5.2 Hybrid pixel detectors 24

6. References 26

During the past decade charge-coupled device (CCD) detectors have increasingly become the preferred choice of medium for recording data in the electron microscope. The CCD detector itself can be likened to a new type of television camera with superior properties, which makes it an ideal detector for recording very low exposure images. The success of CCD detectors for electron microscopy, however, also relies on a number of other factors, which include its fast response, low noise electronics, the ease of interfacing them to the electron microscope, and the improvements in computing that have made possible the storage and processing of large images.

CCD detectors have already begun to be routinely used in a number of important biological applications such as tomography of cellular organelles (reviewed by Baumeister, 1999), where the resolution requirements are relatively modest. However, in most high- resolution microscopic applications, especially where the goal of the microscopy is to obtain structural information at near-atomic resolution, photographic film has continued to remain the medium of choice. With the increasing interest and demand for high-throughput structure determination of important macromolecular assemblies, it is clearly important to have tools for electronic data collection that bypass the slow and tedious process of processing images recorded on photographic film.

In this review, we present an analysis of the potential of CCD-based detectors to fully replace photographic film for high-resolution electron crystallographic applications.

1. Introduction 29

2. Landmarks in NMR of macromolecules 32

2.1 Protein structures and methods development 32

2.1.1 Sequential assignment method 32

2.1.2 Triple-resonance experiments 34

2.1.3 Structures of large proteins 36

2.2 Protein–nucleic acid complexes 37

2.3 RNA structures 38

2.4 Membrane-bound systems 39

3. NMR spectroscopy today 40

3.1 State-of-the-art structure determination 41

3.2 New methods 44

3.2.1 Residual dipolar couplings 44

3.2.2 Direct detection of hydrogen bonds 44

3.2.3 Spin labeling 45

3.2.4 Segmental labeling 46

3.3 Protein complexes 47

3.4 Mobility studies 50

3.5 Determination of time-dependent structures 52

3.6 Drug discovery 53

4. The future of NMR 54

4.1 The ease of structure determination 54

4.2 The ease of making recombinant protein 55

4.3 Post-translationally modified proteins 55

4.4 Approaches to large and/or membrane-bound proteins 56

4.5 NMR in structural genomics 56

4.6 Synergy of NMR and crystallography in protein structure determination 56

5. Conclusion 57

6. Acknowledgements 57

7. References 57

Since the publication of the first complete solution structure of a protein in 1985 (Williamson et al. 1985), tremendous technological advances have brought nuclear magnetic resonance spectroscopy to the forefront of structural biology. Innovations in magnet design, electronics, pulse sequences, data analysis, and computational methods have combined to make NMR an extremely powerful technique for studying biological macromolecules at atomic resolution (Clore & Gronenborn, 1998). Most recently, new labeling and pulse techniques have been developed that push the fundamental line-width limit for resolution in NMR spectroscopy, making it possible to obtain high-field spectra with better resolution than ever before (Dötsch & Wagner, 1998). These methods are facilitating the study of systems of ever-increasing complexity and molecular weight.

1. Introduction 68

2. Ferredoxin reduction by photosystem I 72

3. Ferredoxins 73

4. Ferredoxin[ratio ]thioredoxin reductase 73

4.1 Spectroscopic investigations of FTR 76

4.2 The three-dimensional structure of FTR from the cyanobacterium Synechocystis sp. PCC6803 77

4.2.1 The variable subunit 77

4.2.2 The catalytic subunit 81

4.2.3 The iron–sulfur center and active site disulfide bridge 82

4.2.4 The dimer 84

4.3 Thioredoxin f and m 85

4.4 Ferredoxin and thioredoxin interactions 86

4.5 Mechanism of action 88

4.6 Comparison with other chloroplast FTRs 92

5. Target enzymes 95

5.1 NADP-dependent malate dehydrogenase 95

5.1.1 Regulatory role of the N-terminal extension 97

5.1.2 Regulatory role of the C-terminal extension 99

5.1.3 Thioredoxin interactions 101

5.2 Fructose-1,6-bisphosphatase 101

5.3 Redox regulation of chloroplast target enzymes 103

6. Conclusion 103

7. Acknowledgements 104

8. References 104

A pre-requisite for life on earth is the conversion of solar energy into chemical energy by photosynthetic organisms. Plants and photosynthetic oxygenic microorganisms trap the energy from sunlight with their photosynthetic machinery and use it to produce reducing equivalents, NADPH, and ATP, both necessary for the reduction of carbon dioxide to carbohydrates, which are then further used in the cellular metabolism as building blocks and energy source. Thus, plants can satisfy their energy needs directly via the light reactions of photosynthesis during light periods. The situation is quite different in the dark, when these organisms must use normal catabolic processes like non-photosynthetic organisms to obtain the necessary energy by degrading carbohydrates, like starch, accumulated in the chloroplasts during daylight. The chloroplast stroma contains both assimilatory enzymes of the Calvin cycle and dissimilatory enzymes of the pentose phosphate cycle and glycolysis. This necessitates a strict, light-sensitive control that switches between assimilatory and dissimilatory pathways to avoid futile cycling (Buchanan, 1980, 1991; Buchanan et al. 1994; Jacquot et al. 1997; Schürmann & Buchanan, 2000).