Arg e Lys Gl Ag Gin Ala GIn Gly ll Glu e Aa Lys Se 8 Figure 5 MALDI mass spectrum d dissoh crystal of B-recombinse demonstrated that the crystallization pariod. The data indate removal of the C-larminal portion of the protein to yield crystallins product with a frayed end that pannel residues 126136. Figure reproduced from 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 21
PHASING Extraction of electron density and structural information from X-ray diffraction data of protein crystals requires knowledge of the magnitudes and phase angles of the diffracted X-rays The diffraction data itself contains only the magnitudes the associated phases must be obtained by other means 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 22 PHASING Extraction of electron density and structural information from X-ray diffraction data of protein crystals requires knowledge of the magnitudes and phase angles of the diffracted X-rays. The diffraction data itself contains only the magnitudes; the associated phases must be obtained by other means
Available "phasing methods are isomorphe replacement and molecular replacement ous techniques In isomorphic replacement, a heavy atom is introduced into the protein with minimal perturbation of the native protein fold Phasing information is obtained from X-ray diffraction of a crystal of heavy-atom derivatized protein Heavy atoms used for phasing range in size from selenium to uranium 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 23 Available “phasing” methods are isomorphous replacement and molecular replacement techniques. In isomorphic replacement, a heavy atom is introduced into the protein with minimal perturbation of the native protein fold. Phasing information is obtained from X-ray diffraction of a crystal of heavy-atom derivatized protein. Heavy atoms used for phasing range in size from selenium to uranium
MODEL BUILDING AND REFINEMENT After the electron density has been determined from an x-ray diffraction study of a protein a model of the protein structure is built. One question that frequently arises during model building is whether a stretch of sequence that is not observed in the electron density map is either disordered or altogether missing in the protein crystal (perhaps as a result of degradation over time) 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 24 MODEL BUILDING AND REFINEMENT After the electron density has been determined from an X-ray diffraction study of a protein, a model of the protein structure is built. One question that frequently arises during model building is whether a stretch of sequence that is not observed in the electron density map is either disordered or altogether missing in the protein crystal (perhaps as a result of degradation over time)
intramolecular protein cross-linking in combination with mass spectrometry provided restraint information that is potentially useful for protein model building Limited proteolysis, in conjunction with mass spectrometry, 0/27/2005 Chaoqun Wu, Fudan University
10/27/2005 Chaoqun Wu, Fudan University Chaoqun Wu, Fudan University 25 intramolecular protein cross-linking in combination with mass spectrometry provided restraint information that is potentially useful for protein model building Limited proteolysis, in conjunction with mass spectrometry