2BFB

X-RAY DIFFRACTION

Initial Refinement Model(s)
Type	Source	Accession Code	Details
experimental model	PDB	1OLS	PDB ENTRY 1OLS

Crystalization Experiments
ID	Method	pH	Temperature	Details
1	VAPOR DIFFUSION	5.5	295	CRYSTALS WERE GROWN AT 22C VIA THE VAPOR DIFFUSION METHOD IN COMPLEX WITH A 40 AMINO ACID PEPTIDE DERIVED FROM THE SUBUNIT BINDING DOMAIN (SBD) OF THE E2 COMPONENT OF BRANCHED CHAIN ALPHA-KETOACID DEHDROGENASE. THIS COMPLEX WAS FORMED BY MIXING N-TERMINALLY HIS6-TAGGED PROTEIN WITH C-TERMINALLY HIS6-TAGGED SBD IN 50 MM NA-HEPES, PH 7.5, 150 MM KCL, 20 MM DTT AND 5% (V/V) GLYCEROL AT A MOLAR RATIO OF 1:4. CRYSTALS OF THE COMPLEX (20 MG/ML) WERE OBTAINED BY MIXING 3 MICROLITERS OF PROTEIN WITH 3 MICROLITERS OF CRYSTALLIZATION SOLUTION (10% (V/V) POLYETHYLENE GLYCOL 4000, 10% (V/V) MPD AND 0.1M SODIUM CITRATE, PH 5.8) WITH 1 ML OF CRYSTALLIZATION SOLUTION IN THE RESERVOIR. MANGANESE IONS WERE USED INSTEAD OF MAGNESIUM REQUIRED FOR THE BINDING OF THIAMIN DIPHOSPHATE TO THE ENZYME. THE PRESENCE OF MANGANESE IONS IN THE CRYSTALS RESULTED IN IMPROVED X-RAY DIFFRACTION QUALITIES WITHOUT AFFECTING THE CATALYTIC PROPERTIES. CRYSTALS WERE CRYO-PROTECTED BY STEP-WISE TRANSFER INTO CRYO-BUFFER (CRYSTALLIZATION SOLUTION CONTAINING 5-10%(V/V) GLYCEROL).

Crystal Properties
Matthews coefficient	Solvent content
2.5	53.4

Symmetry
Space Group	P 31 2 1

Diffraction Experiment
ID #	Crystal ID	Scattering Type	Data Collection Temperature	Detector	Detector Type	Details	Collection Date	Monochromator	Protocol
1	1	x-ray	100	CCD	CUSTOM		2004-04-12	M	SINGLE WAVELENGTH

Radiation Source
ID #	Source	Type	Wavelength List	Synchrotron Site	Beamline
1	SYNCHROTRON	APS BEAMLINE 19-ID		APS	19-ID

Overall
ID #	Resolution (High)	Resolution (Low)	Percent Possible (Observed)	R Merge I (Observed)	Net I Over Average Sigma (I)	Redundancy	Number Reflections (All)	Number Reflections (Observed)	Observed Criterion Sigma (F)	Observed Criterion Sigma (I)	B (Isotropic) From Wilson Plot
1	1.77	26.77	99.7	0.09	19.4	6.5		82758		-3	18.88

Highest Resolution Shell
ID #	Resolution (High)	Resolution (Low)	Percent Possible (All)	Percent Possible (Observed)	R Merge I (Observed)	Mean I Over Sigma (Observed)	Redundancy	Number Unique Reflections (All)
1	1.77	1.8	99.9		0.74	2	4.4

Statistics
Diffraction ID	Structure Solution Method	Cross Validation method	Starting model	Resolution (High)	Resolution (Low)	Number Reflections (Observed)	Number Reflections (R-Free)	Percent Reflections (Observed)	R-Factor (Observed)	R-Work	R-Free	R-Free Selection Details	Mean Isotropic B
X-RAY DIFFRACTION	MOLECULAR REPLACEMENT	THROUGHOUT	PDB ENTRY 1OLS	1.77	30	81272	1455	99.7	0.145	0.145	0.184	RANDOM	17.05

Temperature Factor Modeling
Anisotropic B[1][1]	Anisotropic B[1][2]	Anisotropic B[1][3]	Anisotropic B[2][2]	Anisotropic B[2][3]	Anisotropic B[3][3]
0.44	0.22		0.44		-0.66

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	34.248
r_dihedral_angle_4_deg	17.679
r_dihedral_angle_3_deg	14.15
r_dihedral_angle_1_deg	5.792
r_scangle_it	4.603
r_scbond_it	2.917
r_angle_refined_deg	1.672
r_mcangle_it	1.657
r_mcbond_it	0.954
r_nbtor_refined	0.312

RMS Deviations
Key	Refinement Restraint Deviation
r_dihedral_angle_2_deg	34.248
r_dihedral_angle_4_deg	17.679
r_dihedral_angle_3_deg	14.15
r_dihedral_angle_1_deg	5.792
r_scangle_it	4.603
r_scbond_it	2.917
r_angle_refined_deg	1.672
r_mcangle_it	1.657
r_mcbond_it	0.954
r_nbtor_refined	0.312
r_nbd_refined	0.217
r_symmetry_hbond_refined	0.207
r_symmetry_vdw_refined	0.186
r_xyhbond_nbd_refined	0.151
r_chiral_restr	0.131
r_bond_refined_d	0.019
r_gen_planes_refined	0.009
r_bond_other_d
r_angle_other_deg
r_gen_planes_other
r_nbd_other
r_nbtor_other
r_xyhbond_nbd_other
r_metal_ion_refined
r_metal_ion_other
r_symmetry_vdw_other
r_symmetry_hbond_other
r_symmetry_metal_ion_refined
r_symmetry_metal_ion_other
r_mcbond_other
r_mcangle_other
r_scbond_other
r_scangle_other
r_long_range_B_refined
r_long_range_B_other
r_rigid_bond_restr
r_sphericity_free
r_sphericity_bonded