CRYSTAL PACKING ANALYSIS AND ANALYTICAL SIZE-EXCLUSION CHROMATOGRAPHY SUPPORT THE ASSIGNMENT OF A MONOMER AS THE SIGNIFICANT OLIGOMERIC FORM IN SOLUTION.
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Components
#1: Protein
Putativemonooxygenase
Mass: 25422.156 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Fusobacterium nucleatum subsp. nucleatum (bacteria) Strain: ATCC 25586 / Gene: FN1347 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: Q8RDZ4
Mass: 18.015 Da / Num. of mol.: 41 / Source method: isolated from a natural source / Formula: H2O
Sequence details
THE CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH ...THE CONSTRUCT WAS EXPRESSED WITH A 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: 2.23 Å3/Da / Density % sol: 44.86 %
Crystal grow
Temperature: 277 K / Method: vapor diffusion, sitting drop / pH: 5.6 Details: 0.2000M NH4OAc, 30.0000% PEG-4000, 0.1M Citrate pH 5.6, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 277K
Type: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Dec 3, 2009 / 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
0.97925
1
3
0.97894
1
Reflection
Resolution: 2.55→29.607 Å / Num. obs: 14638 / % possible obs: 97.9 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 53.814 Å2 / Rmerge(I) obs: 0.078 / Net I/σ(I): 9
Reflection shell
Resolution (Å)
Rmerge(I) obs
Mean I/σ(I) obs
Num. measured obs
Num. unique obs
Diffraction-ID
% possible all
2.55-2.64
0.654
1.3
5184
2693
1
95.7
2.64-2.75
0.457
1.9
5705
2946
1
99
2.75-2.87
0.333
2.6
5333
2740
1
98.6
2.87-3.02
0.241
3.5
5515
2838
1
99
3.02-3.21
0.168
5.1
5480
2819
1
98.5
3.21-3.46
0.101
7.9
5529
2834
1
98.1
3.46-3.8
0.07
11.3
5315
2727
1
97.8
3.8-4.35
0.05
15
5511
2817
1
97.4
4.35-5.46
0.036
19.1
5454
2783
1
97.7
5.46-29.607
0.028
22
5634
2875
1
97.7
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Phasing
Phasing
Method: MAD
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Processing
Software
Name
Version
Classification
NB
REFMAC
5.5.0102
refinement
PHENIX
refinement
SHELX
phasing
MolProbity
3beta29
modelbuilding
XSCALE
datascaling
PDB_EXTRACT
3.006
dataextraction
XDS
datareduction
SHELXD
phasing
autoSHARP
phasing
Refinement
Method to determine structure: MAD / Resolution: 2.55→29.607 Å / Cor.coef. Fo:Fc: 0.937 / Cor.coef. Fo:Fc free: 0.904 / Occupancy max: 1 / Occupancy min: 0.5 / SU B: 30.986 / SU ML: 0.301 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.358 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 SELENOMETHIONIN INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE ...Details: 1.HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS.2.A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONIN INCORPORATION DURING PROTEIN EXPRESSION. THE OCCUPANCY OF THE SE ATOMS IN THE MSE RESIDUES WAS REDUCED TO 0.75 TO ACCOUNT FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. 3 .ACETATE (ACT) FROM THE CRYSTALLIZATION WAS MODELED INTO THE STRUCTURE 4.ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY. 4. ASN 90 ON BOTH THE A AND B CHAINS ARE FLAGGED AS MOLPROBITY RAMACHANDRAN OUTLIERS, AND IT IS LIKELY THAT THIS IS RELATED TO DISORDERED ELECTRON DENSITIES AT THIS RESIDUE.
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.261
1470
10.1 %
RANDOM
Rwork
0.212
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-
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obs
0.217
14626
99.44 %
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Solvent computation
Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: BABINET MODEL WITH MASK
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