CRYSTAL STRUCTURE OF AN URONATE ISOMERASE (BH0493) FROM BACILLUS HALODURANS C-125 AT 2.00 A RESOLUTION

Joint Center for Structural Genomics (JCSG)

Crystal structure of uncharacterized protein (NP_241359.1) from Bacillus halodurans at 2.00 A resolution

Single crystal Si(111) bent (horizontal focusing)

G(MSE)SINSREVLAEKVKNAVNNQPVTD(MSE)HTHLFSPNFGEILLWDIDELLTYHYLVAEV(MSE)RWTDVSIEAFW
A(MSE)SKREQADLIWEELFIKRSPVSEACRGVLTCLQGLGLDPATRDLQVYREYFAKKTSEEQVDTVLQLANVSDVV
(MSE)TNDPFDDNERISWLEGKQPDSRFHAALRLDPLLNEYEQTKHRLRDWGYKVNDEWNEGSIQEVKRFLTDWIER
(MSE)DPVY(MSE)AVSLPPTFSFPEESNRGRIIRDCLLPVAEKHNIPFA(MSE)(MSE)IGVKKRVHPALGDAGDFVGK
AS(MSE)DGVEHLLREYPNNKFLVT(MSE)LSRENQHELVVLARKFSNL(MSE)IFGCWWF(MSE)NNPEIINE(MSE)T
R(MSE)R(MSE)E(MSE)LGTSFIPQHSDARVLEQLIYKWHHSKSIIAEVLIDKYDDILQAGWEVTEEEIKRDVADLFSR
NFWRFVGRNDHVTSVKVEQQT

GMSINSREVLAEKVKNAVNNQPVTDMHTHLFSPNFGEILLWDIDELLTYHYLVAEVMRWTDVSIEAFWAMSKREQADLIW
EELFIKRSPVSEACRGVLTCLQGLGLDPATRDLQVYREYFAKKTSEEQVDTVLQLANVSDVVMTNDPFDDNERISWLEGK
QPDSRFHAALRLDPLLNEYEQTKHRLRDWGYKVNDEWNEGSIQEVKRFLTDWIERMDPVYMAVSLPPTFSFPEESNRGRI
IRDCLLPVAEKHNIPFAMMIGVKKRVHPALGDAGDFVGKASMDGVEHLLREYPNNKFLVTMLSRENQHELVVLARKFSNL
MIFGCWWFMNNPEIINEMTRMRMEMLGTSFIPQHSDARVLEQLIYKWHHSKSIIAEVLIDKYDDILQAGWEVTEEEIKRD
VADLFSRNFWRFVGRNDHVTSVKVEQQT

THE STRUCTURE WAS SOLVED USING COMBINATION OF MOLECULAR REPLACEMENT (1J5S) AND MULTIPLE-WAVELENGTH ANOMALOUS DISPERSION METHODS. INITIAL HEAVY ATOM SITES WERE OBTAINED USING DIFFERENCE FOURIER WITH MR AND DENSITY MODIFICATION PHASES.

NANODROP, 7.5% PEG 8000, 36.0% 2-methyl-2,4-pentanediol, 0.1M Sodium cacodylate pH 6.33, VAPOR DIFFUSION, SITTING DROP, temperature 277K

BIOMOLECULE: 1,2,3,4
THIS ENTRY CONTAINS THE CRYSTALLOGRAPHIC ASYMMETRIC UNIT
WHICH CONSISTS OF 12 CHAIN(S). SEE REMARK 350 FOR
INFORMATION ON GENERATING THE BIOLOGICAL MOLECULE(S).
SIZE EXCLUSION CHROMATOGRAPHY WITH STATIC LIGHT
SCATTERING SUPPORTS THE ASSIGNMENT OF A TRIMER AS A
BIOLOGICALLY SIGNIFICANT OLIGOMERIZATION STATE.

SEQUENCE
THE CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG
MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV
PROTEASE LEAVING ONLY A GLYCINE, FOLLOWED BY THE
TARGET SEQUENCE.

1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.
2. ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY.
3. 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.
4. THREE UNIDENTIFIED DENSITY BLOBS ARE MODELLED AS UNKNOWN
LIGANDS (UNL). EACH UNL CONTAINS A HEAVY ATOM (ARSENIC) AND
RANDOMLY PLACED OXYGEN ATOMS.
5. GOL, FMT, CL, NA, ACT AND PO4 ARE FROM BUFFER OR
CRYSTALLIZATION/CRYO SOLVENT.
6. THE TEST SET FOR FREE-R CALCULATION IS SELECTED BY
THIN SHELL METHOD.
7. THE RAMACHANDRAN OUTLIERS IN RESIDUE 41 ARE SUPPORTED
BY DENSITY.

SIZE EXCLUSION CHROMATOGRAPHY WITH STATIC LIGHT SCATTERING SUPPORTS THE ASSIGNMENT OF A TRIMER AS A BIOLOGICALLY SIGNIFICANT OLIGOMERIZATION STATE.
URONATE ISOMERASE, STRUCTURAL GENOMICS, JOINT CENTER FOR STRUCTURAL GENOMICS, JCSG, PROTEIN STRUCTURE INITIATIVE, PSI-2, ISOMERASE

MSINSREVLAEKVKNAVNNQPVTDMHTHLFSPNFGEILLWDIDELLTYHYLVAEVMRWTDVSIEAFWAMSKREQADLIWE
ELFIKRSPVSEACRGVLTCLQGLGLDPATRDLQVYREYFAKKTSEEQVDTVLQLANVSDVVMTNDPFDDNERISWLEGKQ
PDSRFHAALRLDPLLNEYEQTKHRLRDWGYKVNDEWNEGSIQEVKRFLTDWIERMDPVYMAVSLPPTFSFPEESNRGRII
RDCLLPVAEKHNIPFAMMIGVKKRVHPALGDAGDFVGKASMDGVEHLLREYPNNKFLVTMLSRENQHELVVLARKFSNLM
IFGCWWFMNNPEIINEMTRMRMEMLGTSFIPQHSDARVLEQLIYKWHHSKSIIAEVLIDKYDDILQAGWEVTEEEIKRDV
ADLFSRNFWRFVGRNDHVTSVKVEQQT