CRYSTAL PACKING ANALYSIS AND SIZE-EXCLUSION CHROMATOGRAPHY COUPLED WITH STATIC LIGHT SCATTERING SUPPORT THE ASSIGNMENT OF A TRIMER AS THE SIGNIFICANT OLIGOMERIC FORM IN SOLUTION.
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Components
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Protein , 1 types, 1 molecules A
#1: Protein
putativepeptidase / Protein containing aminopeptidase domain
Mass: 50586.395 Da / Num. of mol.: 1 / Mutation: P309S Source method: isolated from a genetically manipulated source Source: (gene. exp.) Clostridium acetobutylicum (bacteria) / Gene: CA_C2195 / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: Q97H19
Mass: 18.015 Da / Num. of mol.: 221 / Source method: isolated from a natural source / Formula: H2O
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Details
Sequence details
THIS CONSTRUCT WAS EXPRESSED WITH A PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH ...THIS 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. DNA SEQUENCING OF THE CLONED CONSTRUCT SHOWS A SERINE AT POSITION 309 INSTEAD OF A PROLINE. THE SERINE AT POSITION 309 IS SUPPORTED BY THE ELECTRON DENSITY.
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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: 3.79 Å3/Da / Density % sol: 67.52 %
Resolution: 2.37→29.062 Å / Num. obs: 31178 / % possible obs: 99.9 % / Redundancy: 5.5 % / Biso Wilson estimate: 38.179 Å2 / Rmerge(I) obs: 0.189 / Rsym value: 0.189 / Net I/σ(I): 9
Reflection shell
Rmerge(I) obs: 0.01 / Diffraction-ID: 1
Resolution (Å)
Redundancy (%)
Mean I/σ(I) obs
Num. measured all
Num. unique all
Rsym value
% possible all
2.37-2.43
5.6
0.8
12651
2267
1.017
100
2.43-2.5
5.6
0.8
12501
2234
0.907
100
2.5-2.57
5.6
1
12116
2175
0.775
100
2.57-2.65
5.6
1.1
11775
2106
0.68
100
2.65-2.74
5.6
1.3
11386
2039
0.57
100
2.74-2.83
5.6
1.6
11049
1977
0.487
100
2.83-2.94
5.6
1.9
10596
1897
0.398
100
2.94-3.06
5.6
2.3
10293
1845
0.323
100
3.06-3.2
5.6
3
9886
1777
0.254
100
3.2-3.35
5.5
3.7
9328
1681
0.204
100
3.35-3.53
5.5
4.6
8952
1617
0.162
100
3.53-3.75
5.5
5.7
8490
1533
0.126
100
3.75-4.01
5.5
6.5
7882
1431
0.109
100
4.01-4.33
5.5
7.4
7413
1343
0.095
100
4.33-4.74
5.5
8.6
6864
1244
0.082
100
4.74-5.3
5.5
8.2
6179
1128
0.083
100
5.3-6.12
5.4
7.6
5372
987
0.091
100
6.12-7.49
5.4
8
4593
858
0.085
100
7.49-10.6
5.2
9.4
3479
669
0.064
100
10.6-29.06
4.8
8.6
1780
370
0.064
95.3
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Phasing
Phasing
Method: MAD
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Processing
Software
Name
Version
Classification
NB
REFMAC
5.5.0053
refinement
PHENIX
refinement
SHELX
phasing
MolProbity
3beta29
modelbuilding
SCALA
3.2.5
datascaling
PDB_EXTRACT
3.006
dataextraction
MOSFLM
datareduction
SHELXD
phasing
autoSHARP
phasing
Refinement
Method to determine structure: MAD / Resolution: 2.37→29.062 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.935 / Occupancy max: 1 / Occupancy min: 0.4 / SU B: 11.776 / SU ML: 0.122 / TLS residual ADP flag: LIKELY RESIDUAL / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.201 / ESU R Free: 0.18 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES Details: 1.HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. 2.ATOM RECORDS CONTAIN 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 RECORDS CONTAIN 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 FOR THE REDUCED SCATTERING POWER DUE TO PARTIAL S-MET INCORPORATION. 4.ZINC (ZN) HAS BEEN MODELED AT THE PUTATIVE ACTIVE SITE BASED ON ITS PRESENCE AS A CO-CRYSTALLIZATION COMPOUND AND ANOMALOUS DIFFERENCE FOURIER MAP. 5.CHLORIDE (CL), IMIDAZOLE (IMD) AND (4R)-2-METHYLPENTANE-2,4-DIOL (MRD) FROM THE CRYSTALLIZATION SOLUTION HAVE BEEN MODELED IN THE SOLVENT STRUCTURE. 6.THE RAMACHANDRAN OUTLIER AT RESIDUE PRO190 IS SUPPORTED BY ELECTRON DENSITY.
Rfactor
Num. reflection
% reflection
Selection details
Rfree
0.212
1576
5.1 %
RANDOM
Rwork
0.169
-
-
-
obs
0.171
31177
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: MASK
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