CRYSTAL PACKING ANALYSIS AND ANALYTICAL SIZE EXCLUSION CHROMATOGRAPHY COUPLED WITH STATIC LIGHT SCATTERING SUPPORT THE ASSIGNMENT OF A TRIMER AS A SIGNIFICANT OLIGOMERIC FORM IN SOLUTION.
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Components
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Protein , 1 types, 1 molecules A
#1: Protein
Putativearomatic-ringhydroxylatingdioxygenase / Probable Ring hydroxylating alpha subunit
Mass: 46487.980 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Ruegeria sp. TM1040 (bacteria) / Gene: benA, TM1040_3219 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: Q1GMC3
Mass: 18.015 Da / Num. of mol.: 469 / Source method: isolated from a natural source / Formula: H2O
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Details
Sequence details
THIS CONSTRUCT WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS ...THIS CONSTRUCT WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY THE TARGET SEQUENCE.
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Experimental details
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Experiment
Experiment
Method: X-RAY DIFFRACTION / Number of used crystals: 1
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Sample preparation
Crystal
Density Matthews: 2.85 Å3/Da / Density % sol: 56.85 %
Crystal grow
Temperature: 277 K / Method: vapor diffusion, sitting drop / pH: 8.29 Details: 23.8000% polyethylene glycol monomethyl ether 2000, 0.0100M nickel (II) chloride, 0.1M TRIS pH 8.29, NANODROP', VAPOR DIFFUSION, SITTING DROP, temperature 277K
Type: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Feb 10, 2010 / Details: Flat mirror (vertical focusing)
Radiation
Monochromator: Single crystal Si(111) bent monochromator (horizontal focusing) Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
ID
Wavelength (Å)
Relative weight
1
0.91837
1
2
0.97949
1
3
0.97895
1
Reflection
Resolution: 1.8→28.155 Å / Num. obs: 48265 / % possible obs: 99.3 % / Observed criterion σ(I): -3 / Redundancy: 6.31 % / Biso Wilson estimate: 22.737 Å2 / Rmerge(I) obs: 0.06 / Net I/σ(I): 14.33
Reflection shell
Resolution (Å)
Rmerge(I) obs
Mean I/σ(I) obs
Num. measured obs
Num. unique obs
% possible all
1.8-1.86
0.577
2.16
28099
8783
98.9
1.86-1.94
0.421
3
32555
10184
99.1
1.94-2.03
0.274
4.4
30765
9585
99.2
2.03-2.13
0.199
6.1
28346
8822
99.2
2.13-2.27
0.14
8.4
32011
9942
99.3
2.27-2.44
0.1
11.4
29634
9189
99.4
2.44-2.69
0.076
14.8
31247
9675
99.5
2.69-3.07
0.052
20.7
29995
9290
99.6
3.07-3.87
0.034
31.4
30874
9574
99.7
3.87-28.155
0.024
40.4
30919
9582
99.5
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Phasing
Phasing
Method: MAD
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Processing
Software
Name
Version
Classification
NB
REFMAC
5.6.0073
refinement
PHENIX
refinement
SHELX
phasing
MolProbity
3beta29
modelbuilding
XSCALE
datascaling
PDB_EXTRACT
3.006
dataextraction
XDS
datareduction
autoSHARP
phasing
Refinement
Method to determine structure: MAD / Resolution: 1.8→28.155 Å / Cor.coef. Fo:Fc: 0.965 / Cor.coef. Fo:Fc free: 0.951 / Occupancy max: 1 / Occupancy min: 0.3 / SU B: 3.589 / SU ML: 0.063 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.098 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: 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 ...Details: 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. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 4. NICKEL (NI), CHLORIDE (CL) AND TROMETHAMINE (TRS; TRIS) FROM THE CRYSTALLIZATION SOLUTION AND GLYCEROL (GOL) FROM THE CRYOPROTECTANT SOLUTION HAVE BEEN MODELED. 5. AN IRON-SULPHUR [2FE-2S] CLUSTER HAS BEEN MODELED. 6. THE PRESENCE OF IRON AND NICKEL HAVE BEEN VERIFIED BY X-RAY FLUORESCENCE AND BY CALCULATING ANOMALOUS DIFFERENCE FOURIER MAPS FROM DATA COLLECTED BELOW AND ABOVE THE IRON AND NICKEL K-SHELL ABSORPTION EDGES.
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.186
2418
5 %
RANDOM
Rwork
0.154
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-
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obs
0.156
48240
99.98 %
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Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
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