2. Cloning, expression and purification Obtaining protein samples for crystallization trials and NMR measurements likely will be the bottleneck in the structural genomics process Although the current methods are sufficiently powerful to begin the project, entirely new technologies may arise, such as an in vitro system that already has allowed researchers to produce proteins up to concentrations of 6 mg/ml Shigeyuki Yokoyama, Tokyo University) 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 11 2. Cloning, expression and purification Obtaining protein samples for crystallization trials and NMR measurements likely will be the bottleneck in the structural genomics process. Although the current methods are sufficiently powerful to begin the project, entirely new technologies may arise, such as an in vitro system that already has allowed researchers to produce proteins up to concentrations of 6 mg / ml (Shigeyuki Yokoyama, Tokyo University). Obtaining protein samples for crystallization trials and NMR measurements likely will be the bottleneck in the structural genomics process. Although the current methods are sufficiently powerful to begin the project, entirely new technologies may arise, such as an in vitro system that already has allowed researchers to produce proteins up to concentrations of 6 mg / ml (Shigeyuki Yokoyama, Tokyo University)
3. Crystallization and X-ray crystallography Crystals have a higher chance of being formed when the protein species in solution is Conformationally homogeneous. This can be evaluated by dynamic light scattering, as well as by limited proteolysis combined with mass spectroscopy. For example, candidates that are monodisperse under standard aqueous conditions have a probability of at least 75% to yield crystals suitable for X-ray studies, in comparison to 10% for poly-disperse samples 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 12 3. Crystallization and X-ray crystallography Crystals have a higher chance of being formed when the protein species in solution is Conformationally homogeneous. This can be evaluated by dynamic light scattering, as well as by limited proteolysis combined with mass spectroscopy. For example, candidates that are monodisperse under standard aqueous conditions have a probability of at least 75% to yield crystals suitable for X-ray studies, in comparison to 10% for poly-disperse samples. Crystals have a higher chance of being formed when the protein species in solution is Conformationally homogeneous. This can be evaluated by dynamic light scattering, as well as by limited proteolysis combined with mass spectroscopy. For example, candidates that are monodisperse under standard aqueous conditions have a probability of at least 75% to yield crystals suitable for X-ray studies, in comparison to 10% for poly-disperse samples
Protein Expression, Isolation Purification Protein Domain Crystallization Elucidation Phase Determination Model Building 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 13
Atomic resolution structure determinations of proteins by X ay crystallography require protein construct design, expression and purification ecrystallization trials, phase determination model building 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 14 Atomic resolution structure determinations of proteins by Xray crystallography require: •protein construct design, •expression and purification, •crystallization trials, •phase determination, •model building
Mass Spectrometry can be used to 1. verify that the desired protein construct has been correctly expressed 2. to define compact domains in the target protein 3. to assess the components contained within the protein crystals, 4. to screen for successful incorporation of seleno methionine and other heavy metal reagents used for phasing 5. to address issues of modeling topology, and side chain proximity 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 15 Mass Spectrometry can be used to 1. verify that the desired protein construct has been correctly expressed, 2. to define compact domains in the target protein, 3. to assess the components contained within the protein crystals, 4. to screen for successful incorporation of selenomethionine and other heavy metal reagents used for phasing. 5. to address issues of modeling, topology, and sidechain proximity