Structural Biochemistry/Proteins/Protein Structure determination methods
There are many methods that can be used to determine protein structure.
X-ray crystallography is an experimental technique that is used to determine the structure of a molecule. X-ray crystallography works because the x-ray radiation used to study the sample is of a wavelength that is short enough to be able to discern the features of the molecule. The sample of interest is isolated, purified, and crystallized, and then the crystal sample has a beam of x-ray radiation fired at it. The path of the x-ray photons is perturbed depending on the structure of the molecules making up the structure of the crystal, and the path of the x-rays is captured on a photosensitive paper behind the crystal sample. The patterns the x-rays make on the photosensitive paper are analyzed and the structure of the molecule can be deduced.
Recombinant Protein Refolding Methods used in conjunction with X-Ray Crystallography and NMR:
Because x-ray crystallography and NMR require large amounts (on the order of milligrams) of a purified protein (often unattainable with complications in current purification techniques) to analyze the protein’s structure, recombinant techniques are usually employed whereby a host organism is manipulated to express the protein to be studied. Usually, protein refolding methods must then be used because the protein does not fold properly and abnormalities, known as inclusion bodies, in the protein’s structure develop. To attain the correctly folded protein, the abnormalities are removed; the protein is denatured and then refolded to its correct structure. As a result, the refolded protein can then be studied via x-ray crystallography or NMR. Structure and function analysis of known proteins confirms that this method is comparable to studying the protein directly from its native source.
A solution of proteins is placed in a magnetic field and the effects of different radio frequencies on the resonance of different atoms in a protein are measured. Proteins must be small (~120 residues) and must be soluble for this method. Ab initio methods, homology modeling, and fold recognition are also other popular methods used to determine the structure of tertiary proteins.
A visualization can be created by this method which takes place at or below liquid nitrogen temperatures. It is a fairly new technique that can create visualizations at an extremely high resolution. This method is a form of electron microscopy which utilizes the extremely low temperatures to reduce the occurrence of radiation damage to the specimen.
Tertiary structure prediction
Ab initio methods is used to predict tertiary structure of protein from first principle. It bases on Thermodynamic hypothesis predicts that the native conformation of a protein corresponds to a global free energy minimum of the protein/solvent system. Homology modeling is a class of methods for constructing an atomic-resolution model of a protein from its amino acid sequence. Its motivation is if sequence similarity is high, then structural similarity is probably high, too. Almost all homology modeling techniques rely on the identification of one or more known protein structures likely to resemble the structure of the query sequence, and on the production of an alignment that maps residues in the query sequence to residues in the template sequence. The sequence alignment and template structure are then used to produce a structural model of the target. Because protein structures are more conserved than DNA sequences, detectable levels of sequence similarity usually imply significant structural similarity.
Hard X-ray Fluourescence Tomography
Hard X-ray fluorescence (XRF) microscopy is a powerful method for structural visualization. It detects traces of metal distributions in biological systems like Cu and Zn. 10-15 elements are mapped at the same time, which leads to accurate elemental colocalisation maps. The trace elements are important for most life forms. Metals play an essential role in many proteins by catalyzing functions or for a structural role. Metals are also recognized to have an impact on human health and disease.
X-ray fluorescence is suited for quantifying trace elements. X-ray fluorescence can be excited through x-ray beams or through exposures to a particle like a proton or electron. This helps map the elemental content at high spatial resolution. X-ray microprobes have zone-plates, Kirkpatrick-Baez mirrors, compound refractive lenses, and tapered capillaries, with ranging probe sizes and photo intensities.
Tomography (known as slice imaging) is a two-dimensional technique that is performed on many neighboring slices together, resulting in a three dimensional reconstruction. XRF has also used full-field imaging and structure detector approaches. The technologies have mixed strengths and weaknesses with varied spatial resolutions, sensitivities, and different elemental contrast. Projection tomography uses projections of a specimen as input data to a tomographic reconstruction algorithm. However, x-ray fluorescence micrographs are not exactly equivalent to projection imaging because of self-absorption effects, which include the absorption of the incident beam and re-absorption of the fluorescence by the specimen. Heavier metals in thicker biological tissue have successfully used back projection without correction. Confocal tomography is a direct-space approach to scanning XRF tomography with axial resolution reaching below 5 micrometers. A collimator or lens confines the field of view of the energy-dispersive detector so that the signal derives from only a small portion of the illuminated column. The probe volume is then reduced to a spheroid and elemental distribution is mapped by scanning the specimen through the probe volume in three dimensions. The confocal geometry also allows direct access to a small region of specimen; however, it can be very difficult to target features of interest.
Self-absorption plays a significant role in XRF tomography, and so good correction algorithms are required to ensure image fidelity to expand the specimen size domain, to extend the elemental domain, and to maintain quantitative data accuracy.
Soft X-ray Tomography
In soft x-ray tomography, projection images of a specimen are collected at different angles around a rotation or “tilt” axis, which is mathematically capable of being computed to reconstruct the specimen. A problem that is encountered with three-dimensional tomography is that biological materials become damaged when exposed to intense light in an x-ray microscope from photons or from ultra violet illumination in a fluorescence microscope. However, with soft x-ray tomography, collections of images are acquired at 1-2 degree increments through 180-degree rotation. This causes the specimen to get structurally damaged by receiving a large dose of radiation. However, cryoimmobilization of the specimen avoids this problem. When cells are imaged at liquid nitrogen temperature, more than a thousand soft x-ray projection images can be collected without apparent signs of radiation damage.
The major advantage of water-window soft x-ray tomography is that it can be applied to any imaging investigation in cell biology such as imaging simple bacteria, to yeast and algae, to advanced eukaryotic cells and tissues. The images also keep eukaryotic cells in the native state without the use of stains or labels, which is something that can’t be done with light or electron microscopy.
Electron Tomography, also known as ET, is a novel visualization technique that does what other structure determination methods cannot do in that this method is able to bridge the gap between the atomic structural information of supramolecular structures and its cellular events. It allows visualization of not only the structure of a molecule but its association with other structures and organelles. For instance, electron tomography has opened doors to the observation of virus propagation and viral life cycle in the host cell. Tomography slices allow the examination of molecular architecture of virus as it attaches to cells, penetrate cells, move to replication sites, assemble progeny and transport them to the membranous regions, and exits the cell. Though this technique is still new, it has already been applied to human and simian immunodeficiency viruses (HIV and SIV) and other viruses that infect plant, animals, and humans.
The following are examples of cellular events that have been observed and elucidated through the use of Electron Tomography:
1) Virus Entry: ET shows the presence of "entry claws" which are characteristic architectures of virus coming into contact with the host cell. The entry claw is made up of five to seven rods and represents the interaction between the viral spikes and cell surface receptors. Furthermore, CT shows that some cells, such as vaccinia virus infected cells, shows clear change in shape of the vaccinia virus before and after intrusion of the cell.
2) Virus Factory: ET sheds more light on viroplasms, which are inclusion bodies found at the viral replication site responsible for virus assembly and replication. ET supported the discovery of core proteins P1, P3, P5, and P7 inside the viroplasm and outer capsid proteins P2, P8, and P9, and the formation of virus particles around the viroplasm. With this information, scientists were able to propose a three-step viral replication process: 1)formation of core particles inside the viroplasm, 2) core particle coating with outer capsids at the periphery of the viroplasms, and 3) transportation of mature virus to the membrane region by microtubules.
3) Transportation of Virions inside the Cell: The third step of the proposed viral replication process was observed and confirmed by ET. After the virus propagate at the initial site, they move to another site for secondary multiplication. This movement to another site occurs through virus utilization filamentous substances such as microfilaments and microtubules (MTs) composed of actin and beta tubulins. ET has allowed visualization of the mechanism of cytosolic transportation and strong suggestion that microtubules aid in the transportation of newly assembled virus to the membrane which leads to cell-to-cell spread of the virus. This suggests that microtubules assist in cell-to-cell spread rather than in the entry of viruses to the replication site. Finally, ET shows a gap between the virus and the microtubules indicating that the virus particles do not interact directly with microtubules. Instead, the gap is filled with a rod-like structure that might act as a plus end-directed motor.
In conclusion, ET has shown to have two advantages to the other structure determining methods. The first is that ET prevents misleading conclusions based on 2D structures. Second, observation of finer structures in cells and virus allow elucidation of the organization of detailed virus particles in association with microtubules. This has only been observed using ET.
Soft X-ray Microscopy
In soft x-ray microscopes, third generation synchrotrons are used as the x-ray source. Soft x-ray projection images are a result of precision nanostructured x-ray lenses, high-efficiency direct detection CCD cameras, and well designed transmission X-ray microscopes. Images are generated using phase contrast techniques.
Soft x-ray microscopes are operated using photons with energies in the “water window,” which is the region of the spectrum that lies between the K shell absorption edges of carbon and oxygen. White cellular water remains transparent as the structures in the cell are visualized as a function of biochemical composition and density. Lipid droplets which are structures that absorb more than high water content organelles like vacuoles.
Electron Tomography of the supramolecular structure of virus-infected cells. Kenji Iwasaki and Toshihiro Omura.