The chances of obtaining crystals of sufficient quality and quantity to allow determination of three-dimensional protein structures using synchrotron radiation are significantly increased using a mix of robots geared to different crystallization techniques.
That is the conclusion of a screening study by researchers in Japan, led by Seiki Kuramistu of RIKEN’s SPring-8 Center in Harima, recently reported in Acta Crystallographica (1).
The work was part of the whole-cell project on the bacterium Thermus thermophilus HB8 (Fig. 1), which is found naturally in hot springs at temperatures of up to 85 °C. The aim of the project is to increase understanding of cells at a molecular level by determining the structures and functions of all proteins encoded by genes. Thermus was chosen as a model organism because it has a minimal set of genes which codes for about 2,000 proteins which are highly stable for analysis and more than 70% of which have human equivalents.
The standard means of determining protein structure, x-ray crystallography, involves aligning protein molecules into a lattice of repeating series of ‘unit cells’, and then passing x-rays through the resulting crystal. The structure of the protein is ‘solved’ by analyzing the resulting diffraction pattern.
But proteins are of irregular shape and the protein lattice is held together only by relatively weak electrostatic forces. So protein crystals are generally fragile and highly sensitive to environmental conditions. These must be adjusted to optimum levels for crystallization. At best it takes several hours to grow crystals suitable for data collection, but typically it takes months. Thus, protein crystallization has proved a major bottleneck in the whole-cell project.
In an attempt to increase efficiency, the researchers used 18 sample proteins from Thermus to test the capabilities of robots which use different techniques to crystallize proteins—sitting-drop vapor diffusion, hanging-drop vapor diffusion and a modified microbatch technique. They also trialed a microfluidic device designed to rapidly determine the best initial conditions, but which could not produce crystals in large enough quantities for diffraction.
The research team found that both vapor diffusion robots produced diffraction-quality crystals quicker than the microbatch robot—the sitting-drop being the faster. The microbatch robot, however, was most likely to be successful. The microfluidic device outperformed the other three on both counts. On the basis of these results the researchers used a combination of a sitting-drop and a microbatch robot to successfully determine structures for 360 of 944 purified proteins for the whole-cell project.
1. Iino, H., Naitow, H., Nakamura, Y., Nakagawa, N., Agari, Y., Kanagawa, M., Ebihara, A., Shinkai, A., Sugahara, M., Miyano, M., et al. Crystallization screening test for the whole-cell project on Thermus thermophilus HB8. Acta Crystallographica F64, 487–491 (2008).
Win-win strategies for climate and food security
02.10.2017 | International Institute for Applied Systems Analysis (IIASA)
The personality factor: How to foster the sharing of research data
06.09.2017 | ZBW – Leibniz-Informationszentrum Wirtschaft
University of Maryland researchers contribute to historic detection of gravitational waves and light created by event
On August 17, 2017, at 12:41:04 UTC, scientists made the first direct observation of a merger between two neutron stars--the dense, collapsed cores that remain...
Seven new papers describe the first-ever detection of light from a gravitational wave source. The event, caused by two neutron stars colliding and merging together, was dubbed GW170817 because it sent ripples through space-time that reached Earth on 2017 August 17. Around the world, hundreds of excited astronomers mobilized quickly and were able to observe the event using numerous telescopes, providing a wealth of new data.
Previous detections of gravitational waves have all involved the merger of two black holes, a feat that won the 2017 Nobel Prize in Physics earlier this month....
Material defects in end products can quickly result in failures in many areas of industry, and have a massive impact on the safe use of their products. This is why, in the field of quality assurance, intelligent, nondestructive sensor systems play a key role. They allow testing components and parts in a rapid and cost-efficient manner without destroying the actual product or changing its surface. Experts from the Fraunhofer IZFP in Saarbrücken will be presenting two exhibits at the Blechexpo in Stuttgart from 7–10 November 2017 that allow fast, reliable, and automated characterization of materials and detection of defects (Hall 5, Booth 5306).
When quality testing uses time-consuming destructive test methods, it can result in enormous costs due to damaging or destroying the products. And given that...
Using a new cooling technique MPQ scientists succeed at observing collisions in a dense beam of cold and slow dipolar molecules.
How do chemical reactions proceed at extremely low temperatures? The answer requires the investigation of molecular samples that are cold, dense, and slow at...
Scientists from the Max Planck Institute of Quantum Optics, using high precision laser spectroscopy of atomic hydrogen, confirm the surprisingly small value of the proton radius determined from muonic hydrogen.
It was one of the breakthroughs of the year 2010: Laser spectroscopy of muonic hydrogen resulted in a value for the proton charge radius that was significantly...
17.10.2017 | Event News
10.10.2017 | Event News
10.10.2017 | Event News
18.10.2017 | Materials Sciences
18.10.2017 | Physics and Astronomy
18.10.2017 | Physics and Astronomy