#228 - Dec 2018 Directed Evolution of Enzymes similarity (5)
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Assembly
Deposited unit
A: Mlr3007 protein B: Mlr3007 protein C: Mlr3007 protein D: Mlr3007 protein E: Mlr3007 protein F: Mlr3007 protein G: Mlr3007 protein H: Mlr3007 protein hetero molecules
Mass: 18.015 Da / Num. of mol.: 773 / Source method: isolated from a natural source / Formula: H2O
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Details
Sequence details
THIS CONSTRUCT (RESIDUES 1-220) WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG ...THIS CONSTRUCT (RESIDUES 1-220) 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: 2.59 Å3/Da / Density % sol: 52.49 %
Crystal grow
Temperature: 277 K / Method: vapor diffusion, sitting drop / pH: 6.2 Details: 0.200M NH4I, 20.00% PEG-3350, No Buffer pH 6.2, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 277K
Monochromator: DOUBLE CRYSTAL MONOCHROMATOR SI(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
Wavelength: 0.97915 Å / Relative weight: 1
Reflection
Resolution: 2.3→29.683 Å / Num. obs: 91682 / % possible obs: 98.6 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 32.589 Å2 / Rmerge(I) obs: 0.084 / Net I/σ(I): 8.97
Reflection shell
Resolution (Å)
Rmerge(I) obs
Mean I/σ(I) obs
Num. measured obs
Num. unique obs
Diffraction-ID
% possible all
2.3-2.38
0.428
1.8
31555
16526
1
96.2
2.38-2.48
0.368
2.1
35432
18456
1
99.1
2.48-2.59
0.289
2.7
32567
16924
1
99
2.59-2.73
0.231
3.5
34708
18000
1
99.2
2.73-2.9
0.179
4.3
33396
17283
1
99
2.9-3.12
0.124
6.3
33224
17167
1
99.1
3.12-3.43
0.075
9.9
33570
17324
1
99
3.43-3.93
0.047
15.6
34196
17612
1
98.7
3.93-4.93
0.032
21.3
33564
17222
1
98.7
4.93-29.683
0.031
22
34409
17591
1
97.9
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Phasing
Phasing
Method: SAD
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Processing
Software
Name
Version
Classification
NB
MolProbity
3beta29
modelbuilding
PDB_EXTRACT
3.1
dataextraction
SHELXD
phasing
autoSHARP
phasing
XSCALE
datascaling
REFMAC
5.5.0110
refinement
XDS
datareduction
Refinement
Method to determine structure: SAD / Resolution: 2.3→29.683 Å / Cor.coef. Fo:Fc: 0.936 / Cor.coef. Fo:Fc free: 0.909 / Occupancy max: 1 / Occupancy min: 0.25 / SU B: 11.129 / SU ML: 0.15 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.312 / ESU R Free: 0.228 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY. 3. A MET-INHIBITION PROTOCOL WAS USED FOR SELENOMETHIONINE INCORPORATION DURING PROTEIN ...Details: 1. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2. ATOM RECORD CONTAINS RESIDUAL B FACTORS ONLY. 3. 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 TO ACCOUNT FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. 4. CHLORIDE (CL) AND IODIDE (IOD) IONS FROM THE PURIFICATION AND CRYSTALLIZATION SOLUTIONS HAVE BEEN MODELED INTO THE STRUCTURE. BOTH HALIDES BIND IN SIMILAR ENVIRONMENTS AND MANY OF THE STIES LIKELY CONTAIN A MIXTURE OF I- AND CL- WITH INCOMPLETE OCCUPANCY. OCCUPANCIES WERE ESTIMATED WITH REFINEMENT IN PHENIX.REFINE. IODIDES WERE MODELED AT POSITIONS SUPPORTED BY ANOMALOUS DIFFERENCE FOURIERS PEAKS AND SITES THAT PHENIX.REFINE REFINED OCCUPANCIES GREATER THAN 1.1 WHEN MODELED AS CHLORIDE. 5. ETHYLENE GLYCOL (EDO), USED AS A CRYOPROTECTANT AND POLYETHYLENE GLYCOL (PEG) FROM THE CRYSTALLIZATION SOLUTION WERE MODELED INTO THE STRUCTURE. 6. ELECTRON DENSITY SUPPORTS THE MODELING OF A BOUND PYRIDOXAL-5'-PHOSPHATE (PLP) COFACTOR ON EACH SUBUNIT AS N'-PYRIDOXYL-LYSINE-5'-MONOPHOSPHATE (LLP), IN WHICH THE PLP COFACTOR IS COVALENTLY BOUND TO LYS 117. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 8. TLS GROUPS WERE ASSIGNED WITH THE AID OF THE TLSMD SERVER.
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.2407
4592
5 %
RANDOM
Rwork
0.1932
-
-
-
obs
0.1956
91628
99.63 %
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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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