CRYSTAL PACKING ANALYSIS AND ANALYTICAL SIZE EXCLUSION CHROMATOGRAPHY SUPPORT THE ASSIGNMENT OF A MONOMER AS A SIGNIFICANT OLIGOMERIZATION STATE IN SOLUTION.
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Components
#1: Protein
HeterogeneousnuclearribonucleoproteinL / hnRNP L
Mass: 24164.891 Da / Num. of mol.: 1 / Fragment: RRM 3 domain containg residues 376-586 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Mus musculus (house mouse) / Gene: BC027206, Hnrnpl, Hnrpl / Plasmid: SpeedET / Production host: Escherichia Coli (E. coli) / Strain (production host): HK100 / References: UniProt: Q8R081
Mass: 18.015 Da / Num. of mol.: 126 / Source method: isolated from a natural source / Formula: H2O
Sequence details
THE CONSTRUCT WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS ...THE CONSTRUCT WAS EXPRESSED WITH AN N-TERMINAL PURIFICATION TAG MGSDKIHHHHHHENLYFQG. THE TAG WAS REMOVED WITH TEV PROTEASE LEAVING ONLY A GLYCINE (0) FOLLOWED BY RESIDUES 376-586 OF THE TARGET SEQUENCE. NUMBERING IS BASED ON THE UNIPROTKB Q8R081 VERSION 2 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: 2.14 Å3/Da / Density % sol: 42.41 %
Crystal grow
Temperature: 277 K / Method: vapor diffusion, sitting drop / pH: 7.5 Details: 10.00% 2-propanol, 20.00% polyethylene glycol 4000, 0.1M HEPES pH 7.5, NANODROP, VAPOR DIFFUSION, SITTING DROP, temperature 277K
Resolution: 2.15→29.512 Å / Num. obs: 11314 / % possible obs: 98.1 % / Observed criterion σ(I): -3 / Biso Wilson estimate: 26.714 Å2 / Rmerge(I) obs: 0.091 / Net I/σ(I): 6.8
Reflection shell
Resolution (Å)
Rmerge(I) obs
Mean I/σ(I) obs
Num. measured obs
Num. unique obs
Diffraction-ID
% possible all
2.15-2.23
0.452
1.9
4232
2242
1
96.5
2.23-2.32
0.351
2.4
4108
2144
1
99
2.32-2.42
0.297
2.9
3935
2048
1
100
2.42-2.55
0.249
3.2
4206
2195
1
98.9
2.55-2.71
0.204
3.8
4231
2196
1
99
2.71-2.92
0.148
5.3
4162
2156
1
99.2
2.92-3.21
0.107
7
4083
2115
1
98.2
3.21-3.67
0.062
11.1
4107
2126
1
97.5
3.67-4.61
0.046
14.6
4065
2108
1
97.5
4.61-29.512
0.04
16.1
4141
2137
1
95.7
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Phasing
Phasing
Method: MAD
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Processing
Software
Name
Version
Classification
NB
MolProbity
3beta29
modelbuilding
PDB_EXTRACT
3.1
dataextraction
SHELX
phasing
SHARP
phasing
XSCALE
December6, 2010
datascaling
BUSTER-TNT
2.8.0
refinement
XDS
datareduction
SHELXD
phasing
BUSTER
2.8.0
refinement
Refinement
Method to determine structure: MAD / Resolution: 2.15→29.512 Å / Cor.coef. Fo:Fc: 0.9523 / Cor.coef. Fo:Fc free: 0.9262 / Occupancy max: 1 / Occupancy min: 0.4 / Cross valid method: THROUGHOUT / σ(F): 0 Details: 1. 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 ...Details: 1. 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. 2. ATOM RECORD CONTAINS SUM OF TLS AND RESIDUAL B FACTORS. ANISOU RECORD CONTAINS SUM OF TLS AND RESIDUAL U FACTORS. 3. WATERS WERE EXCLUDED FROM AUTOMATIC TLS ASSIGNMENT. 4. 2-PROPANOL (IPA) AND GLYCEROL (GOL) MOLECULES FROM THE CRYSTALLIZATION/CRYOPROTECTION SOLUTIONS ARE MODELED. 5. THE MAD PHASES (HL COEFFICIENTS) WERE USED AS RESTRAINTS DURING REFINEMENT.
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