The foundations of electron crystallography and cryo-electron microscopy (cryo-EM) were laid in the early 1970s. The first structural determination of a biological unstained crystalline specimen was on Bacteriorhodopsin in native purple membranes [1]. This breakthrough required an understanding of the affects of drying on delicate samples, whereby glucose embedding was applied and the limitations from beam damage were overcome using low-dose techniques at the microscope [2]. Taylor and Glaeser [3] used electron diffraction of frozen, hydrated protein crystals and also recorded images of samples preserved in a frozen hydrated state [4]. The cryo-EM technique was further developed by the pioneering work of Dubochet and co-workers at the EMBL, where liquid ethane was used as a cryogen to immobilize specimens in a layer of vitreous ice [5].

Structural biology of membrane proteins is of central importance in cellular biology and for the development of new drugs. Membrane proteins represent the majority of today’s drug targets in pharmaceutical research. Enormous progress has been made in recent years in the structure determination of membrane proteins, whereby the majority of structures were determined by X-ray crystallography. Our databases now contain more than 400 unique folds of membrane proteins (http://​blanco.​biomol.​uci.​edu/​mpstruc), and this number is growing almost exponentially. A remaining challenge is the understanding of the dynamic function of membrane proteins in the lipid bilayer. Here, electron crystallography can provide significant insight into the conformation of membrane proteins in the membrane-embedded form. Two-dimensional membrane protein crystals are stabilizing the membrane proteins in a lipid bilayer, and the surfaces of the proteins are readily accessible to ligands, changing buffer conditions, or binding partners.

Electron crystallography studies the structure of membrane proteins in a two-dimensional crystalline arrangement in a phospholipid bilayer membrane. The atomic models of tubulin [6] and of the membrane proteins BR [7], LHCII [8], and water channels [9–13], were among the first protein structures determined by electron crystallography at atomic resolution. Several other membrane proteins classified as transporters, ion pumps, receptors, and membrane-bound enzymes have been studied at slightly lower resolution allowing the localization of secondary structure motifs such as transmembrane helices [14].

Electron crystallography can be used to study membrane protein structures at resolutions of 3 Å or better (e.g., [11, 15, 16]), demonstrating the value of this approach. Electron crystallography represents an alternative method for structure determination, when fragile membrane protein complexes cannot be grown into 3D protein crystals for X-ray diffraction or are not available in sufficient quantities for NMR measurements. Information gleaned from EM reconstructions complement X-ray diffraction data in several ways. EM preparations are often considered more physiologically relevant compared to crystals formed in the presence of detergents, as is sometimes the case with X-ray crystallography. Membrane-inserting proteins that undergo conformational changes between the soluble and the membrane-inserted states are likely to be best studied by electron crystallography. 2D membrane crystals are frequently grown easier than 3D crystals of membrane proteins and offer to the membrane proteins a more native environment than most 3D crystal forms. Membrane crystals are also advantageous for the structure determination of co-crystals, when preformed crystals are to be incubated with protein binding partners. Cryo-EM also offers the possibility to freeze-capture various protein conformational states as freezing rates in ethane ~10⁶ °C/s [17] outperform the transient isomeric states (milliseconds) of proteins [18].

Technological advancements like the availability of coherent intermediate voltage electron sources and nitrogen- or helium-cooled and stable sample stages [19] allow recording of high-resolution data of biological macromolecules. Improvement in detection devices such as CCD and CMOS cameras allows efficient data recording of electron diffraction patterns [20], and the advent of the first direct electron detection devices (DED) is likely to revolutionize also the imaging of 2D crystals. Advancements in sample preparation with the sandwich-back-injection technique using non-wrinkled carbon films [21–23] and the spot-scanning data collection method [24] strongly reduce the resolution-limiting charge effect during data acquisition of tilted samples. Direct electron detectors show promise to also reduce beam-induced and mechanical sample drift when using the so called dose-fractionation or movie mode.

2D membrane protein crystallization usually requires detergent-solubilized and detergent-purified protein typically at a concentration of 1–2 mg/mL. Crystallization can be achieved by various methods [25–30]. So far, the best-ordered 2D membrane protein crystals were obtained after slow and controlled detergent dialysis in the presence of added phospholipids [26]. However, several other 2D crystallization methods were also developed [30–34].