The dual advantages of high resolution and fast throughput make synchrotron-based x-ray crystallography, like that which is being carried out at Berkeley Lab's Advanced Light Source, the undisputed mainstay for solving protein structures. However, some 20 to 40 percent of all proteins are extremely difficult or even impossible to crystallize, including many found in the membranes which control the transportation of molecules and communication of signals across cell surfaces. This means that other technologies will also have critical roles to play.
Tools for Investigating Nanoscale Machines
Physical Biosciences investigators, in concert with other Berkeley Lab investigators, are developing novel, complementary combinations of established and newly developed microscopy tools for macromolecular analysis. Four of these tools are: atomic force microscopy (AFM), cryo-electron microscopy, single-molecule fluorescence microscopy (SFM), and optical tweezers microscopy. All of these methods rely on single molecule analysis and provide complementary, structural, and dynamic biological information.
AFM is a descendent of scanning tunneling microscopy (STM). Images are generated by recording the deflection of a very sharp tip as it is dragged over a sample. In this way, an atom by atom picture of the surface is taken.
Cryo-electron microscopy is a relatively new technique that ensures preservation of a biological sample and accuracy of the images, compared to standard electron microscopy. Samples are prepared by rapidly freezing them, and are then preserved in an aqueous-like vacuum environment. There are few artifacts, staining is not required, and radiation damage is minimal.
Single molecule fluorescence (SMF) is a new type of biological imaging technique
where light emitted from individual fluorophores (e.g. quantum dots) is captured and used to investigate the conformational dynamics of proteins and nuclic acids.
Optical tweezers microscopy is a relatively new tool (~ late 80's and early 90's) that
permits molecule-by-molecule measurement and manipulation of a sample. Through the use of a highly controlled laser beam, the mechanical properties of DNA are measured by grabbing the DNA at both ends and pulling it, thus revealing its resistance to stretching.
Currently, we are refining these technolgies (e.g. by improving their temporal or spatial resolution), as well as developing entirely new "hybrid" instruments by combining AFM with SMF, and optical tweezers with SMF. The goal of the latter effort (tentatively called "Initiative for Advanced Instrumentation") is to develop and construct hybrid instruments that will allow scientists to mechanically manipulate molecular machines (using AFM or optical tweezers) while simultaneously watching the machine respond to the external perturbation using SMF. Such instrumentation will not only benefit efforts to understand biological machines, but also assist in the construction of entirely new machines and devices from scratch, the focus of the Molecular Foundry program at Berkeley.
Electron Crystallography and Single-Particle Electron Cryo-Microscopy
The cell has a modular architecture in which many central processes are performed by specialized molecular machines. Molecular machines are characterized by complexes of as many as ten or more protein. Each protein contributes a specialized function to the machine, such as recognition, regulation or catalysis. Molecular machines operate in a cyclical fashion and may have many intermediate structural conformations. The Nogales Lab, studies the architecture of large biological complexes and the structural basis for their regulation. They are applying cryo electron microscopy (cryo-Em) and image analysis to characterize the structure and assembly of microtubles and their interaction with cellular factors and antimitotic ligands. The Nogales Lab also uses cryo-em methodology, such as automation of data collection and use of highly parallelized computer architectures to increase research throughput. The electron crystallography and cryo-em research program at LBNL also includes the work of Dr. Kenneth Downing in the Life Sciences Division
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