ANALYTICAL SIZE EXCLUSION CHROMATOGRAPHY WITH STATIC LIGHT SCATTERING SUPPORTS THE ASSIGNMENT OF A DIMER AS A SIGNIFICANT OLIGOMERIZATION STATE IN SOLUTION.
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Components
#1: Protein
Putativeoxidoreductase / / Putative dehydrogenase
Mass: 51228.184 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Rhodopseudomonas palustris (phototrophic) Gene: RPA1076 / Plasmid: SpeedET / Production host: Escherichia coli (E. coli) / Strain (production host): HK100 / References: UniProt: Q6NAV4
Mass: 18.015 Da / Num. of mol.: 919 / 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: 3.75 Å3/Da / Density % sol: 67.19 %
Crystal grow
Temperature: 293 K / Method: vapor diffusion, sitting drop Details: 0.60M potassium dihydrogen phosphate, 25.00% Glycerol, 0.60M sodium dihydrogen phosphate, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 293K
Type: MARMOSAIC 325 mm CCD / Detector: CCD / Date: Jun 10, 2010
Radiation
Monochromator: DOUBLE CRYSTAL SI (111) / Protocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
ID
Wavelength (Å)
Relative weight
1
0.91837
1
2
0.97922
1
3
0.97894
1
Reflection
Resolution: 2.57→49.357 Å / Num. obs: 144716 / % possible obs: 98.9 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 52.656 Å2 / Rmerge(I) obs: 0.114 / Net I/σ(I): 10.26
Reflection shell
Resolution (Å)
Rmerge(I) obs
Mean I/σ(I) obs
Num. measured obs
Num. unique obs
Diffraction-ID
% possible all
2.57-2.66
0.824
1.7
53712
14230
1
99.9
2.66-2.77
0.679
2
56337
14940
1
99.8
2.77-2.89
0.478
2.9
52177
13806
1
99.9
2.89-3.05
0.347
4
57569
15236
1
99.6
3.05-3.24
0.243
5.6
54466
14394
1
99.6
3.24-3.48
0.15
8.7
52764
13946
1
99.2
3.48-3.83
0.101
12.3
55174
14562
1
99.1
3.83-4.38
0.064
18
54805
14442
1
98.5
4.38-5.5
0.049
21.7
55002
14442
1
97.8
5.5-49.357
0.039
25.5
55963
14800
1
96
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Phasing
Phasing
Method: MAD
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Processing
Software
Name
Version
Classification
NB
SHELX
phasing
REFMAC
5.5.0110
refinement
XSCALE
datascaling
PDB_EXTRACT
3.1
dataextraction
XDS
datareduction
SHELXD
phasing
autoSHARP
phasing
Refinement
Method to determine structure: MAD / Resolution: 2.57→49.357 Å / Cor.coef. Fo:Fc: 0.95 / Cor.coef. Fo:Fc free: 0.933 / Occupancy max: 1 / Occupancy min: 0.5 / SU B: 15.762 / SU ML: 0.175 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.346 / ESU R Free: 0.24 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. WATERS WERE EXCLUDED FROM AUTOMATIC TLS ASSIGNMENT. 5.A FLAVIN-ADENINE DINUCLEOTIDE (FAD) COFACTOR WAS MODELED INTO EACH OF THE SIX MONOMERS IN THE ASYMMETRIC UNIT. 6.AN UNKNOWN LIGAND (UNL) WAS MODELED INTO THE PUTATIVE ACTIVE NEAR THE FAD BINDING SITE ON EACH MONOMER 6. PHOSPHATE (PO4) AND POTASSIUM (K) FROM THE CRYSTALLIZATION WERE MODELED INTO THE STRUCTURE. THE OCCUPANCY ON SOME OF THE PHOSPHATES WERE DECREASED TO 0.5 TO ACCOUNT FOR THE DECREASED SCATTERING. 7. UNIDENTIFIED ELECTRON DENSITY NEAR ARG 56, ARG 103,AND HIS 128 ON EACH MONOMER WAS NOT MODELED. 8. THE ELECTRON DENSITIES CORRESPONDING TO ASN 45 ON THE B AND E CHAINS ARE POOR, AND THESE RESIDUES ARE RAMACHANDRAN OUTLIERS IN MOLPROBITY. GLN 146 ON THE F CHAIN CHAIN AND PRO 384 ON THE D AND E CHAINS ARE RAMACHANDRAN OUTLIERS IN MOLPROBITY EVEN THOUGH THEIR MODELING IS SUPPORTED BY ELECTRON DENSITY.
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.2266
7270
5 %
RANDOM
Rwork
0.1931
-
-
-
obs
0.1948
144702
98.91 %
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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: MASK
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