1.Background of research and development
Three-dimensional crystallization of a membrane protein is famous for its difficulty, and is represented as one type of a high-risk, high-return study.
The first crystallization was accomplished for the reaction centers of photosynthetic bacteria by H. Michel from Germany, who was awarded Nobel Prize in Chemistry for his achievements in 1988.
Michel advocated that it would be necessary to prepare a crystal with packing completely different from in vivo by removing phospholipid (Type 2 Crystal, Fig. 1) for crystal analysis of membrane proteins solubilized with a surfactant, and crystals in which proteins naturally buried in a lipid bi-layer membrane are stacked (Type 1 Crystal) might not be good since they would not grow. He also stated that phospholipid might be originally inhomogeneous posing adverse influence on crystallization. His assertion was believed broadly and, so to speak, became a common sense.

Since steam diffusion method was exclusively used for three-dimensional crystallization at the X-ray level, and a method of removing surfactant by dialysis forming lipid bi-layer membrane (two-dimensional crystal) was used for crystallization at the electron microscope level, it was believed that "Crystallization at the X-ray and electron microscope levels are essentially different".

Ca²⁺ pump protein (SERCA1a) in skeletal muscle is naturally present in great quantity, and its purification method had been already established in 1970. Therefore, it's one of few membrane proteins which anyone can crystallize, so to speak, and rumors that crystals were made appeared and disappeared for over 20 years or more duration. Although crystals were obtained at the electron microscope level, no one could remove phospholipid without losing activity or make crystals as large as the X-ray level.

Fig. 1 Packing of three-dimensional crystals of membrane proteins and
three-dimensional crystals of Ca²⁺ pump proteins in various states

2.Achievements
It was in the middle of the 1980s that Toyoshima started to deal with Ca²⁺ pump proteins. At first, a reconstruction method of three-dimensional image of a tube-like crystal by an electron microscope was developed, and the first conformation of uncombined Ca²⁺ was obtained. On the other hand, it succeeded in production of a three-dimensional ultra-thin microcrystalline showing good electron diffraction pattern under Ca²⁺ existence, however, no crystal large enough for X-ray crystal structure analysis could be obtained. However, considering that there might not be essential difference between crystallization at the X-ray and electron microscope levels, Toyoshima decided to investigate extension of the electron microscope level method. Actually, addition of phospholipid was essential to maintain activity during purification in the case of Ca²⁺ pump proteins, and the difference between the aforementioned 2 methods might be only "whether to use a precipitant effectively," was what he considered.

Such trial contrary to the common sense resulted in success in determination of the Ca²⁺ pump crystal structure at the resolution of 2.6 Å which was high resolution as a membrane protein crystal at that time. It was in 2000. This was the first structure determination in a metal ion pump, moreover, Ca²⁺ pump which is the ion most extensively used in biocontrol, thus, it had a very great impact. Furthermore, Toyoshima succeeded in structure determination of 8 intermediate states almost completely covering the reaction cycle of Ca²⁺ pump by using crystallization method using this phospholipid and a metal fluoride complex as an analogue of phosphoric acid (Figs. 1, 2). The result enabled us to understand the outline of the mechanism transporting ions against the concentration gradient from the atomic structure. The magnitude of the structural change revealed by the result which nobody could anticipate, greatly reversed the conventional common sense, for example the large slide movement between transmembrane helice that was believed to be engaged firmly. Furthermore, it was found that the ion pump uses a lipid bi-layer membrane as a part of transportation mechanism, shedding a new light on the physiological role of lipid bi-layer membrane.

Although this crystallization method was the expansion of the two-dimensional crystallization method for an electron microscope, the application to three-dimensional crystallization was not easy. Since there are many factors in crystallization, success will be achieved only after they are set properly. To begin with, it was not even known what factor was an important factor or not. For example, the type of phospholipid (single chain or mixture, percentage of saturation and unsaturated fatty acid, etc.), the quantity ratio of protein: phospholipid: surfactant are important factors which may affect the success of crystallization, thus, systematic investigation was required. Therefore, many ideas for improving repeatability were necessary. In particular, establishment of a quick and simple quantitative method of phospholipid and a surfactant was important.

Fig. 2 Crystal structure of Ca²⁺ pump corresponding to states of
immediately before and after releasing two Ca²⁺ (I, II) confined in
the membrane. A domain rotates around the axis I by 90 degrees.
A ribbon indicates an alpha helix and an arrow indicates a beta sheet.

3.Meaning of the achievements
With the success of this study, other groups came to succeed in crystallization by actively adding phospholipid, and greatly contributed to the expansion of the structural analysis of membrane proteins. Especially, it is very meaningful that it was proved that the structural analysis of membrane proteins originally functioning in a lipid bi-layer membrane may be possible under almost natural condition. Additionally, up to date, there are few proteins including water-soluble ones whose reaction mechanism have been structurally understood in such details.

The design of a drug based on the crystal structure with high resolution may reduce trial and error remarkably, enabling great reduction of time and cost needed for development. It has been said that most targets of future drug design are membrane proteins, especially, structure study of membrane proteins of higher animals is important with respect to medicine and drug design. On the other hand, in the current situation of searching for those can be crystallized by the conventional method and performing structure determination, most of them are from bacteria even if they are related proteins, structure determination of higher animal membrane proteins is quite rare. Active addition of phospholipid may greatly expand the range of membrane proteins which can be crystallized. Therefore, the importance will increase remarkably in the future. Actually, many cases in which crystallization succeeded for the first time by actively introducing phospholipid are being reported.