Novel cell-free protein crystallization methods advancing structural biology

The new cell-free protein crystallization (CFPC) method developed by Tokyo Institute of Technology, including direct protein crystallization, is an important advance in
the field of structural biology.
This technique makes possible
the analysis of unstable proteins that cannot be studied using traditional methods.
Analyzing these will increase our knowledge of
cellular processes and functions.

While we are familiar with certain crystals used in everyday life, such as salt and sugar, there is another group of crystals that are invisible to the naked eye and are essential
for our physiological properties.
Microscopic protein crystals are found in living cells and help maintain processes
such as immune system activation, protein storage and protection.

To better understand the relationship between the structure and function of protein crystals, scientists have developed intracellular protein crystallization (ICPC) methods that allow direct observation of protein crystals in living cells, ensuring high-quality crystals without the need for purification procedures or complex screening methods
.
However, despite its many advantages, few structures have been reported because crystals formed in living cells do not have
the size and quality required for analysis.
Therefore, a group of researchers from Japan led by Professor Takafumi Ueno of the Tokyo Institute of Technology worked to develop a better method
.
Recently, they made a breakthrough!

In their published article, the team reports on the development of a technique that will make the crystallization and analysis of proteins more efficient and effective
.
This technique is a cell-free protein crystallization method (CFPC), which is a mixture of protein crystallization and ICPC in vitro that enables rapid and direct formation of protein crystals without the need for complex crystallization and purification methods
.

ICPC promises to be an important tool for crystal structure analysis, but we need a way to obtain protein crystal structures
with higher resolution.
Therefore, we focus on establishing high-quality protein crystallization with CFPCs, using small-scale and rapid responses," said Professor Ueno, who is also the head of the
Ueno Laboratory at Tokyo Institute of Technology.
Members of the laboratory study naturally occurring protein combinations, their structure and function, with the aim of applying this knowledge to develop innovative biotechnology and energy solutions
.

Going back to the team conducting the current research (some of whom are also members of Ueno's lab), they used a wheat germ protein synthesis kit, a tool for synthesizing polyhedral monomers, a viral protein produced in insect cells by cypovirus infection
.
This protein is then crystallized using the new CFPC method to form nanometer-sized polyhedral crystals (PhCs).

The team was able to complete the process efficiently in 6 hours, using only 20 microliters of reaction mixture
.
Scanning electron microscope images show that PhCs have excellent purity, which allows for a high purity of up to 1.
95 ?? (or 1.
95 angstroms) at a resolution of its structure
.
To further explore the capabilities of their new system, the team performed structural analysis
of crystal-enveloping protein A (CipA).
Its structure was determined at a high resolution of 2.
11 ° , which had never been reported before in this study
.

This work is a huge leap forward in the field of structural biology because it proposes a method that will make the analysis of unstable and low-yield proteins impossible to study
by traditional methods.
This technology is also designed to help develop advanced technologies
for small-scale and rapid protein crystallization and analysis.
Professor Ueno concluded, "The high-quality protein crystals produced with our method will expand the horizons for structure determination and provide us with useful, unprecedented insights
into the complex environment of living cells.
"

Crystal clear view of crystal proteins!