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Current methods in structural proteomics and its applications in biological sciences

View Article: PubMed Central

ABSTRACT

A broad working definition of structural proteomics (SP) is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules. Recently, the process for protein structure determination has become highly automated and SP platforms have been established around the globe, utilizing X-ray crystallography as a tool. Although protein structures often provide clues about the biological function of a target, once the three-dimensional structures have been determined, bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles. This article reviews the current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation.

No MeSH data available.


X-ray data collection facility. a End-station instrumentation at ESRF beamline BM14 (http://www.bm14.eu) illustrating the sample changer used to exchange cryo-frozen crystals on the goniometer and the MARCCD (Marresearch GmbH) detector used to collect diffraction images. The arrow highlights the path of the X-ray beam. b Close-up view showing the frozen crystal sample in the center of the image and the surrounding beamline instrumentation. The red cross and blue circle represent the center and diameter of the X-ray beam on the frozen crystal sample (bottom right)
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Fig5: X-ray data collection facility. a End-station instrumentation at ESRF beamline BM14 (http://www.bm14.eu) illustrating the sample changer used to exchange cryo-frozen crystals on the goniometer and the MARCCD (Marresearch GmbH) detector used to collect diffraction images. The arrow highlights the path of the X-ray beam. b Close-up view showing the frozen crystal sample in the center of the image and the surrounding beamline instrumentation. The red cross and blue circle represent the center and diameter of the X-ray beam on the frozen crystal sample (bottom right)

Mentions: In the past, protein crystals typically ranging in size from tenths of a millimeter to several millimeters were mounted in glass capillary tubes. To collect data, the capillary tube was mounted on a goniometer and exposed to X-rays at room temperature. These X-rays were generated by low flux, sealed tube sources. Nowadays, data collection is handled by automated sample changers and micro-diffractometers in a cryogenic (100 K) environment utilizing brighter synchrotron radiation as the X-ray source. Cryo-freezing the sample inhibits free radicals diffusing through the crystal during data collection: these free radicals cause secondary radiation damage that leads to degradation in the quality of collected data. There are currently in excess of 125 dedicated protein crystallography beamlines around the World. The X-ray films that were used for data recording in the past have now been superseded with charge-coupled devices (CCD) and pixel array detectors, which allow diffraction data to be recorded directly and stored straight to disk. For example, a recent development has been the PILATUS detector (pixel apparatus for the SLS), which has no readout noise, superior signal-to-noise ratio, a readout time of 5 ms and high dynamic range compared to CCD and imaging plate detectors. Delivery of high flux beam at third-generation synchrotron sources coupled with the advances in detector technology and control systems have significantly accelerated the speed of macromolecular diffraction data collection. An example of a state-of-the-art synchrotron X-ray data collection setup is shown in Fig. 5. Nowadays, crystals larger than 50 μm in size can be evaluated at conventional synchrotron beamlines. However, with some targets such as membrane proteins and multi-protein complexes, it is notoriously difficult to obtain crystals of sufficient size and order to generate high-quality diffraction data. Hence, next generation microfocus beamlines with reduced beam sizes have been established at synchrotron sites around the world, allowing measurements to be made on crystals a few micrometers in size. It has been predicted that a complete data set with a signal-to-noise ratio of 2σ at 2 Å resolution could be collectable from a perfect lysozyme crystal measuring just 1.2 μm in diameter using a microfocus beam (Holton and Frankel 2010). A number of crystal structures have been solved using micrometer-sized crystals by merging data from several crystals, including a polyhedron-like protein structure (~5–12 μm) (Coulibaly et al. 2007) and a thermally stabilized recombinant rhodopsin (with crystal dimensions of 5 × 5 × 90 μm3) (Standfuss et al. 2007). Recently, strategies have been developed to determine structures from showers of microcrystals using acoustic droplet ejection (ADE) to transfer 2.5 nl droplets from the surface of microcrystal slurries through the air and onto micromesh loops. Individual microcrystals are located by raster-scanning a several-micron X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets are subsequently merged from several micrometer-sized crystals and this technique has been used to solve 1.8 Ǻ resolution crystal structures (Soares et al. 2011).Fig. 5


Current methods in structural proteomics and its applications in biological sciences
X-ray data collection facility. a End-station instrumentation at ESRF beamline BM14 (http://www.bm14.eu) illustrating the sample changer used to exchange cryo-frozen crystals on the goniometer and the MARCCD (Marresearch GmbH) detector used to collect diffraction images. The arrow highlights the path of the X-ray beam. b Close-up view showing the frozen crystal sample in the center of the image and the surrounding beamline instrumentation. The red cross and blue circle represent the center and diameter of the X-ray beam on the frozen crystal sample (bottom right)
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Related In: Results  -  Collection

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Fig5: X-ray data collection facility. a End-station instrumentation at ESRF beamline BM14 (http://www.bm14.eu) illustrating the sample changer used to exchange cryo-frozen crystals on the goniometer and the MARCCD (Marresearch GmbH) detector used to collect diffraction images. The arrow highlights the path of the X-ray beam. b Close-up view showing the frozen crystal sample in the center of the image and the surrounding beamline instrumentation. The red cross and blue circle represent the center and diameter of the X-ray beam on the frozen crystal sample (bottom right)
Mentions: In the past, protein crystals typically ranging in size from tenths of a millimeter to several millimeters were mounted in glass capillary tubes. To collect data, the capillary tube was mounted on a goniometer and exposed to X-rays at room temperature. These X-rays were generated by low flux, sealed tube sources. Nowadays, data collection is handled by automated sample changers and micro-diffractometers in a cryogenic (100 K) environment utilizing brighter synchrotron radiation as the X-ray source. Cryo-freezing the sample inhibits free radicals diffusing through the crystal during data collection: these free radicals cause secondary radiation damage that leads to degradation in the quality of collected data. There are currently in excess of 125 dedicated protein crystallography beamlines around the World. The X-ray films that were used for data recording in the past have now been superseded with charge-coupled devices (CCD) and pixel array detectors, which allow diffraction data to be recorded directly and stored straight to disk. For example, a recent development has been the PILATUS detector (pixel apparatus for the SLS), which has no readout noise, superior signal-to-noise ratio, a readout time of 5 ms and high dynamic range compared to CCD and imaging plate detectors. Delivery of high flux beam at third-generation synchrotron sources coupled with the advances in detector technology and control systems have significantly accelerated the speed of macromolecular diffraction data collection. An example of a state-of-the-art synchrotron X-ray data collection setup is shown in Fig. 5. Nowadays, crystals larger than 50 μm in size can be evaluated at conventional synchrotron beamlines. However, with some targets such as membrane proteins and multi-protein complexes, it is notoriously difficult to obtain crystals of sufficient size and order to generate high-quality diffraction data. Hence, next generation microfocus beamlines with reduced beam sizes have been established at synchrotron sites around the world, allowing measurements to be made on crystals a few micrometers in size. It has been predicted that a complete data set with a signal-to-noise ratio of 2σ at 2 Å resolution could be collectable from a perfect lysozyme crystal measuring just 1.2 μm in diameter using a microfocus beam (Holton and Frankel 2010). A number of crystal structures have been solved using micrometer-sized crystals by merging data from several crystals, including a polyhedron-like protein structure (~5–12 μm) (Coulibaly et al. 2007) and a thermally stabilized recombinant rhodopsin (with crystal dimensions of 5 × 5 × 90 μm3) (Standfuss et al. 2007). Recently, strategies have been developed to determine structures from showers of microcrystals using acoustic droplet ejection (ADE) to transfer 2.5 nl droplets from the surface of microcrystal slurries through the air and onto micromesh loops. Individual microcrystals are located by raster-scanning a several-micron X-ray beam across the cryocooled micromeshes. X-ray diffraction data sets are subsequently merged from several micrometer-sized crystals and this technique has been used to solve 1.8 Ǻ resolution crystal structures (Soares et al. 2011).Fig. 5

View Article: PubMed Central

ABSTRACT

A broad working definition of structural proteomics (SP) is that it is the process of the high-throughput characterization of the three-dimensional structures of biological macromolecules. Recently, the process for protein structure determination has become highly automated and SP platforms have been established around the globe, utilizing X-ray crystallography as a tool. Although protein structures often provide clues about the biological function of a target, once the three-dimensional structures have been determined, bioinformatics and proteomics-driven strategies can be employed to derive their biological activities and physiological roles. This article reviews the current status of SP methods for the structure determination pipeline, including target selection, isolation, expression, purification, crystallization, diffraction data collection, structure solution, refinement and functional annotation.

No MeSH data available.