Crystal structure of glycosyl hydrolase family 5 (NP_809925.1) from BACTEROIDES THETAIOTAOMICRON VPI-5482 at 2.10 A resolution

Joint Center for Structural Genomics (JCSG)

Single crystal Si(111) bent monochromator (horizontal focusing)

0.20M ammonium sulfate, 40.0% 2-methyl-2,4-pentanediol, 0.1M HEPES pH 7.0, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 293K

THE CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY RESIDUES 22-483 OF THE TARGET SEQUENCE.

(1) HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS (2) A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. (3) AFTER BUILDING THE PROTEIN CHAINS A, B AND C, ANOMALOUS DIFFERENCE MAPS AND DIFFERENCE ELECTRON DENSITY MAPS SUGGESTED THAT THERE WAS A FOURTH SUBUNIT IN THE CRYSTALLOGRAPHIC ASYMMETRIC UNIT. HOWEVER, THE ELECTRON DENSITY FOR THIS SUBUNIT IS POOR, AND BOTH THE ELECTRON DENSITY MAP AND ANOMALOUS DIFFERENCE FOURIER MAPS INDICATE THAT THIS EXTRA SUBUNIT IS DISORDERED. THE ANOMALOUS DIFFERENCE PEAKS WERE USED TO GUIDE THE BUILDING OF CHAIN D. THE PATTERN OF ANOMALOUS DIFFERENCE DENSITY PEAKS SUPPORTS MODELING OF THE SUBUNIT IN TWO HALF OCCUPANCY CONFORMATIONS. NOTE THAT WHILE CHAIN D PART B WOULD SYMMETRY CLASH WITH ITSELF, IT DOES NOT CLASH WITH THE SYMMETRY MATE OF PART A. ADDITIONALLY, CHAIN D PART A DOES NOT CLASH WITH THE SYMMETRY MATE OF CHAIN D PART B. (4) SULFATES (SO4), (4S)-2-METHYL-2,4-PENTANEDIOLS (MPD), AND (4R)-2-METHYL-2,4-PENTANEDIOLS (MRD) FROM THE CRYSTALLIZATION SOLUTION WERE MODELED INTO THE STRUCTURE. (5) ELECTRON DENSITY INDICATES THAT THE PEPTIDE BOND BETWEEN GLY 241 AND HIS 242 ON EACH SUBUNIT IS IN THE CIS CONFIGURATION.

glycosyl hydrolase family 5, Structural Genomics, Joint Center for Structural Genomics, JCSG, Protein Structure Initiative, PSI-2, HYDROLASE