STRUCTURE OF THE CALCIUM-REGULATED PHOTOPROTEIN OBELIN DETERMINED BY SULFUR SAS

Structure of the Ca2+-regulated photoprotein obelin at 1.7 A resolution determined directly from its sulfur substructure.

Preparation and preliminary study of crystals of the recombinant calcium-regulated photoprotein obelin from the bioluminescent hydroid obelia longissima

MSSKYAVKLKTDFDNPRWIKRHKHMFDFLDINGNGKITLDEIVSKASDDICAKLEATPEQTKRHQVCVEAFFRGCGMEYG
KEIAFPQFLDGWKQLATSELKKWARNEPTLIREWGDAVFDIFDKDGSGTITLDEWKAYGKISGISPSQEDCEATFRHCDL
DNSGDLDVDEMTRQHLGFWYTLDPEADGLYGNGVP

STRUCTURE DETERMINED USING THE SULFUR SAS SIGNAL RECORDED AT ID17, APS USING 1.74 ANGSTROM X-RAYS (SEE REMARK 3 REFINEMENT DETAILS)

HANGING DROP VAPOR DIFFUSION USING 5 MICROLITER DROPS CONTAINING EQUAL VOLUMES OF PROTEIN CONCENTRATE (10 MG/ML) AND RESERVOIR SOLUTION: CONTAINING 1.4 M SODIUM CITRATE, PH 6.0., VAPOR DIFFUSION, HANGING DROP, temperature 277K

THE STRUCTURE WAS DETERMINED USING SINGLE WAVELENGTH
ANOMALOUS SCATTERING DATA RECORDED FOR THE NATIVE PROTEIN.
DATA WERE RECORDED ON TWO OBELIN CRYSTALS TO 2.5 ANGSTROMS
RESOLUTION AT BEAMLINE ID-17 (IMCA-CAT), ADVANCED PHOTON
SOURCE (APS), ARGONNE NATIONAL LABORATORY USING A
MARRESEARCH 165 MM CCD DETECTOR AND 1.74 ANGSTROM X-RAYS.
A DATA COLLECTION STRATEGY (HKL2000) WAS DETERMINED FROM
THE INITIAL IMAGE TO GIVE THE STARTING AND ENDING POINTS OF
THE DATA COLLECTION TO INSURE A MINIMUM DATA COMPLETENESS
OF 98 PERCENT. DATA WERE COLLECTED USING THE "FREIDEL
FLIP" METHOD. DATA PROCESSING WAS CARRIED OUT USING
HKL2000. THE PROCESSED DATA FORMING THE TWO DATA SETS WERE
MERGED TO INCREASE REDUNDANCY (WU, ET AL., (2000) J. PROT.
PEP. LETTS. 7, 25-32) AND SOLVE WAS USED TO DETERMINE THE
SIX OF THE EIGHT SULFUR ATOM POSITIONS. PHASES WAS USED
TO REFINE THE SULFUR POSITIONS AND TO LOCATE THE REMAINING
SULFUR SITES BY BIJVOET DIFFERENCE FOURIER ANALYSIS. THREE
ADDITIONAL SITES WERE IDENTIFIED SUGGESTING THAT ONE OF THE
SULFUR SITES WAS MOST PROBABLY AN ANOMALOUS SOLVENT SUCH AS
CHLORIDE. ALL ANOMALOUS SITES WERE TREATED AS SULFUR FOR
THE PHASE CALCULATIONS.
THE NINE SULFUR SITES WERE USED TO ESTIMATE THE INITIAL
PROTEIN PHASES AT 3 ANGSTROM RESOLUTION USING SOLVENT
FLATTENING (ISAS, WANG, (1985) METHODS ENZYMOL. 115, 90
112). A PRELIMINARY CHAIN TRACE WAS DONE USING THE 3.0
ANGSTROM MAP. SEQUENCE ASSIGNMENT WAS BASED ON 8 SULFUR
ATOM POSITIONS. THE MODEL, 99 PERCENT COMPLETE WAS BUILT
USING THE 3.0 ANGSTROM SAS MAP. RESIDUE 1 WAS NOT
OBSERVED IN THE ELECTRON DENSITY AMPS AND IS PRESUMED TO BE
DISORDERED.
THE RESOLUTION OF THE STRUCTURE WAS THEN EXTENDED TO 1.73
ANGSTROMS RESOLUTION DURING THE FINAL STAGES OF REFINEMENT
USING A MERGED DATA SET COLLECTED FROM A SINGLE CRYSTAL
RECORDED AT APS (BEAMLINE ID19, WAVELENGTH 0.94 ANGSTROMS)
AND AT ALS (BEAMLINE 5.0.2, WAVELENGTH 1.0 ANGSTROMS).

BIOLUMINESCENCE, CALCIUM, PHOTOPROTEIN, OBELIN, SULFUR ANOMALOUS SCATTERING, LUMINESCENT PROTEIN