A new cell-free protein crystallisation method developed by the Tokyo Institute of Technology represents a major headway in the field of structural biology

The technique will enable the analysis of unstable proteins that could not be studied using conventional methods. Analysing these will increase our knowledge of cellular processes and functions.

While we are familiar with certain crystals, such as salt and sugar that we use in our everyday life, there is another set of crystals, hidden from the naked eye, that is crucial to our biology.

Microscopic protein crystals are found in living cells and help sustain processes like immune system activation, storage, and protection.

To better understand the relationship between protein crystals’ structure and function, scientists developed the in-cell protein crystallisation (ICPC) method, which can directly observe protein crystals in living cells, ensuring high-quality crystals without the need for purification processes or complex screening methods.

However, despite its many advantages, very few structures were reported because the crystals formed in living cells didn’t have the size and quality that was required for analysis. So, a team of researchers from Japan, led by Professor Takafumi Ueno, head of the Ueno Laboratory at Tokyo Tech, aimed to develop a better method. And recently, they hit a breakthrough!

See also: In defence of fundamental science

Protein crystallisation

The team reported the development of a technique that would make protein crystallisation and analysis more efficient and effective. This technique – a cell-free protein crystallisation (CFPC) method – was a hybrid between in vitro protein crystallisation and ICPC, and allowed rapid and direct formation of crystals without the need for complicated crystallisation and purification methods.

Professor Ueno said: “ICPC is expected to become an important tool in crystal structure analysis, but we need a method to obtain better resolution protein crystal structures. So, we focused on establishing high-quality protein crystallization using CFPC with small-scale and rapid reactions.”

The team conducting the study (some of whom are also members of the Ueno Laboratory) used a wheat germ protein synthesis kit, which is a tool for the synthesis of polyhedrin monomer, a viral protein produced in insect cells by cypovirus infection.

This protein was then crystallised using the new CFPC method, leading to the formation of nano-sized polyhedra crystals (PhCs). The team could efficiently complete this process within six hours, using only 20 microlitres of the reaction mixture.

Scanning electron microscopy images indicated that the PhCs had excellent purity, which allowed the determination of their structure at a resolution as high as 1.95 angstrom (1.95Å).

To further explore the capabilities of their new system, the team carried out the structural analysis of crystalline inclusion protein A (CipA). Its structure was determined at a high resolution of 2.11Å, something that had never been reported before this study.

Big leap forward

This work is a big leap forward in the field of structural biology, as the method it proposes will enable the analysis of unstable and low-yield proteins that cannot be studied via conventional methods.

This technology also aims to aid in the development of advanced techniques for small-scale and rapid crystallisation and analysis.

Ueno concluded: “The high-quality protein crystals produced by our method will expand the horizons of structural determination and provide us with useful and unprecedented insights into the complex environment of living cells.”

The article is published in Scientific Reports.

Image: Schematic illustration of CFPC process using a wheat germ protein synthesis kit to synthesise polyhedrin monomer (PhM) which was further crystallized to nano-sized polyhedra crystals.

Credit: Professor Takafumi Ueno.