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In recent years, the sequencing of the human genome has enabled researchers to understand the full complement of genes that define the blueprint of human life. The next challenge for researchers is to understand the role of the molecules that are encoded in the genes and carry out their instructions. Acting alone or in groups, proteins are the essential active constituents of cells and underlie most of an organism’s biological functions. There are many types of proteins: those that act like motors, moving anything from small cellular particles, to arms and legs; messenger proteins such as hormones that transmit messages to regulate biological processes; those that form machines that either build or break other types of molecules, such as the food that we eat, and transporter proteins that allow select substances to enter and exit the cells.
Understanding of proteins and their role in molecular machines remains a huge challenge. Structural Biology addresses that challenge by focusing on deciphering the three-dimensional shape or “structure” of these molecules, as their shape is intrinsic to their function in the cell.
Each protein has a unique three-dimensional shape, which reveals many of its important properties -- the particular job that it is designed to accomplish, and the sites at which it interacts with other molecules. All proteins are made up of the same building blocks, a linear sequence of 20 different small molecules called amino acids that initially are assembled like beads on a string, but when activated, fold into a stable and well-defined three-dimensional structure. The size of a protein ranges from fewer than one hundred to many thousands of amino acids. The order and number of amino acids in a protein determines how it will fold into a functional state and conversely how mistakes (such as mutations) can cause misfolding that can give rise to disease. When they are folded into their unique shapes, proteins have specific sites that precisely fit partner molecules. This fit is like a lock and key of miniscule proportions. Some proteins join together to form large molecular machines that carry out critical cellular functions like protein synthesis (the ribosome) or mediate transport across membranes (the nuclear pore complex). Often these machines operate smoothly, but when they make mistakes, they set off a cascade of events that can trigger disease. The structural information yielded by the different techniques of Structural Biology enables scientists to understand how molecular machines are formed and how they function. This information is also vital to designing molecular-level therapies and interventions to fix errors that otherwise can cause irreparable harm.
Each of the different technologies of Structural Biology – X-ray Crystallography, Nuclear Magnetic Resonance Spectrometry, and Cryoelectron Microscopy -- provides detailed images of proteins that give scientists insights as to how these critical units function. The images also reveal the flaws resulting from genetic mutations that alter a protein’s structure that can disrupt this critical molecular machinery.
X-ray Crystallography uses intense beams of light, a billion times more powerful than that of the sun, generated by a synchrotron source, to reveal the architecture of molecules in their solid, crystallized form. This well-established technology has resulted in three-dimensional images of thousands of proteins and associated molecules. The first task in X-ray Crystallography is to form crystals from soluble proteins. The crystals are composed of millions of identical protein molecules that form symmetrical lattices. When they are placed in the path of an X-ray beam, the atomic components of the crystal diffract the light and X-rays are scattered in defined directions. The pattern of this scattering provides information about the location of each atom inside the constituent protein molecules. Using computer analysis, this information can be modeled to produce a three-dimensional image of the protein with atomic-level detail. Read more about X-ray Crystallography.
Nuclear Magnetic Resonance Spectrometry (NMR) uses extremely powerful magnets, up to half a million times stronger than the magnetic field of the Earth, to analyze the distance between the atoms that make up a protein. Atoms act as tiny magnets due to the “spin” of their nuclei and when a protein is placed in an NMR, the spins of its’ atoms line up with the field of the powerful NMR magnet much like iron filings in close proximity to a refrigerator magnet. An experiment starts by exposing a protein sample to a pulse of radio waves. This temporarily alters the orientation of individual atoms’ spins which return into their normal position at the end of the pulse, akin to a gust of wind disturbing the branches of a tree. Radio waves emitted during this return reveal the distances between the atoms composing the protein. As with X-ray Crystallography, computers are used to analyze and model this data to produce a three-dimensional image of the protein at the atomic level of resolution. An advantage of NMR over X-ray Crystallography is its ability to create images of proteins in their dynamic state. Read more about NMR spectroscopy.
Cryoelectron Microscopy (CEM) uses electrons to reveal images of either individual molecules or molecular assemblies. The electrons function as light does in a light microscope to illuminate a sample, however the electromagnetic lenses are able to magnify the image of sample up to 1 million fold either on film or on a digital camera. Biological molecules are very sensitive to the electron beam and therefore frozen (cryo) samples are used which do not degrade as quickly as non-frozen samples. Computer programs are required to line-up and average many thousands of individual images to bring out the detail of a molecular structure. Although, most structures are not shown at the atomic level of detail, CEM has the great advantage of being able create images of transient, multi-molecular complexes. When combined with prior knowledge about the constituent proteins from NMR and X-ray Crystallography, this technique has the potential to reveal the specific protein-protein interactions that reveal their functional partnership within the cell. Read more about CEM.