Abstract

Structural and functional information of membrane proteins at ever-increasing resolution is being obtained by electron crystallography. While a large amount of work on the development of methods for electron microscopy and image processing has resulted in tremendous advances in terms of speed of data collection and resolution, general guidelines for crystallization are first starting to emerge. Yet two-dimensional crystallization itself will always remain the limiting factor of this powerful approach in structural biology. Two-dimensional crystallization through detergent removal by dialysis is the most widely used technique. Four main factors need to be considered for the dialysis method: the protein preparation, the detergent, the lipid added as well as any constituent lipid, and the buffer conditions. Equally important is proper and careful screening to identify two-dimensional crystals.

Keywords: Membrane proteins; Electron crystallography; Structure; Two-dimensional crystallization; Electron cryo-microscopy; Cryo-EM; Screening; Lipid-to-protein ratio; Detergent; Image processing

Article Outline

1. Introduction

2. 2D crystallization

2.1. The starting material: protein

2.2. The detergent

2.2.1. Co-purified lipid

2.2.2. The lipid to be added

2.3. The dialysis buffer

2.4. Limitations in the number of parameters for each trial

2.5. Crystallization strategy and starting points

2.6. Observations for designing further experiments

2.7. Testing further crystallization parameters

2.8. Choice of dialysis device

2.9. Storage of crystals

3. Screening samples by electron microscopy

3.1. Grid preparation

3.2. Obtaining an overview: preliminary screening at low magnification

3.3. Screening for crystallinity

3.4. Optical diffraction

3.5. Freeze-fracture

3.6. Assessing the quality of crystals

3.7. Electron cryo-microscopy: imaging and electron diffraction

4. Concluding remarks

Acknowledgements

References