Preparation, Crystallization, and Structure Determination of Chromatin Enzyme/Nucleosome Complexes.

Structural studies of chromatin complexes composed of chromatin factors or enzymes bound to the nucleosome have been constrained by the ability to produce high-quality complexes in the amounts appropriate for biophysical studies and by the difficulty of crystallizing these complexes. We describe here procedures and approaches to prepare chromatin complexes, to crystallize chromatin complexes, and to improve diffraction properties through postcrystallization soaks. Special attention is paid to evaluating the quality of the purified chromatin complexes as well as assessing the presence of the chromatin protein or enzyme in crystals. The methods described for preparing and purifying chromatin complexes should be applicable to biochemical, biophysical, and other structural approaches including cryoelectron microscopy.

Expression, purification, crystallization and preliminary X-ray diffraction studies of recombinant class B non-specific acid phosphatase of Salmonella typhimurium.

The aphA gene of Salmonella enterica sv. Typhimurium strain MD6001 was cloned in the multicopy plasmid pBluescript SK(-). The recombinant AphA protein was purified to homogeneity. The protein crystallized in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 112.4, b = 130.2, c = 139.6 A. Consistent with the self-rotation function, there are two tetramers in the asymmetric unit, indicating a solvent content of approximately 54%. The crystals are composed of biologically active AphA molecules.

Purification, crystallization and preliminary X-ray diffraction studies of recombinant class A non-specific acid phosphatase of Salmonella typhimurium.

The phoN gene of Salmonella enterica sv. Typhimurium strain MD6001 was cloned in the multicopy plasmid pBluescript SK(-). The nucleotide sequence of the cloned gene differs from the corresponding S. typhimurium LT2 sequence at 23 residues, leading to 15 amino-acid differences, but was very close to the S. typhi phoN sequence (only three nucleotide and two amino-acid differences). The recombinant PhoN protein was purified to homogeneity. Two forms of crystals were harvested from a single crystallization condition. Diffraction intensity data were collected using a laboratory X-ray source to resolution limits of 2.5 and 2.8 A for crystals belonging to space group C2 and C222(1), respectively. Based on non-crystallographic symmetry, four monomers of PhoN are expected to be present in the asymmetric unit of the C2 unit cell. Two monomers of a biologically active dimer in the asymmetric unit of the C222(1) unit cell are expected from the Matthews coefficient.