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- PDB-6pqk: Cryogenic crystal structure of barnase A43C/S80C bound to barstar... -

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Basic information

Entry
Database: PDB / ID: 6pqk
TitleCryogenic crystal structure of barnase A43C/S80C bound to barstar C40A/S59C/A67C/C82A
Components
  • Barstar
  • Ribonuclease
KeywordsRNA BINDING PROTEIN / disulfide / Toxin-Antitoxin / complex / nuclease
Function / homology
Function and homology information


Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters / RNA endonuclease activity / RNA binding / extracellular region / cytoplasm
Similarity search - Function
Barstar-like / Barstar (barnase inhibitor) / Barstar (barnase inhibitor) / Barstar-like superfamily / Barnase; Chain D / : / Barnase / Microbial ribonucleases / Guanine-specific ribonuclease N1/T1/U2 / Ribonuclease/ribotoxin ...Barstar-like / Barstar (barnase inhibitor) / Barstar (barnase inhibitor) / Barstar-like superfamily / Barnase; Chain D / : / Barnase / Microbial ribonucleases / Guanine-specific ribonuclease N1/T1/U2 / Ribonuclease/ribotoxin / ribonuclease / Nuclear Transport Factor 2; Chain: A, / Roll / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHATE ION / Ribonuclease / Barstar
Similarity search - Component
Biological speciesBacillus amyloliquefaciens (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.2 Å
AuthorsCaro, J.A. / Djanto, V. / Wand, A.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM102447 United States
CitationJournal: To Be Published
Title: Targeting conformational entropy to modulate binding affinity
Authors: Caro, J.A. / Djanto, V. / Wand, A.J.
History
DepositionJul 9, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 15, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Ribonuclease
C: Ribonuclease
B: Barstar
D: Barstar
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,34612
Polymers49,7184
Non-polymers6288
Water7,566420
1
A: Ribonuclease
D: Barstar
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,2687
Polymers24,8592
Non-polymers4095
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
C: Ribonuclease
B: Barstar
hetero molecules


Theoretical massNumber of molelcules
Total (without water)25,0785
Polymers24,8592
Non-polymers2193
Water362
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)36.704, 68.404, 166.723
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Space group name HallP2ac2ab

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Components

#1: Protein Ribonuclease / Barnase / RNase Ba


Mass: 14522.065 Da / Num. of mol.: 2 / Mutation: A43C, S80C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus amyloliquefaciens (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3)
References: UniProt: P00648, Hydrolases; Acting on ester bonds; Endoribonucleases producing 3'-phosphomonoesters
#2: Protein Barstar / Ribonuclease inhibitor


Mass: 10336.739 Da / Num. of mol.: 2 / Mutation: C40A, S59C, A67C, C82A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus amyloliquefaciens (bacteria) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / References: UniProt: P11540
#3: Chemical
ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: PO4
#4: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C2H6O2
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 420 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.3 Å3/Da / Density % sol: 41.56 %
Crystal growTemperature: 298 K / Method: vapor diffusion, hanging drop / pH: 6.5
Details: 50mM NaPO4 pH 6.5, 25% PEG 8K, 0.1M AmSO4 4uL protein at 10-15 mg/mL in H2O + 4uL motherliquor per hanging drop, over 1 mL motherliquor in well. cryoprotectant used before freezing was: 12. ...Details: 50mM NaPO4 pH 6.5, 25% PEG 8K, 0.1M AmSO4 4uL protein at 10-15 mg/mL in H2O + 4uL motherliquor per hanging drop, over 1 mL motherliquor in well. cryoprotectant used before freezing was: 12.5% glycerol + 12.5% ethylene glycol

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Data collection

DiffractionMean temperature: 80 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSLS-II / Beamline: 17-ID-1 / Wavelength: 0.92013 Å
DetectorType: DECTRIS EIGER X 9M / Detector: PIXEL / Date: Apr 25, 2019
RadiationMonochromator: double crystal Si(111) monochromator with horizontal theta-axis
Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.92013 Å / Relative weight: 1
ReflectionResolution: 1.2→50 Å / Num. obs: 110616 / % possible obs: 83.6 % / Redundancy: 11.7 % / Biso Wilson estimate: 10.91 Å2 / Rmerge(I) obs: 0.083 / Rpim(I) all: 0.024 / Rrim(I) all: 0.087 / Χ2: 0.614 / Net I/σ(I): 5.5 / Num. measured all: 1298292
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
1.2-1.223.60.63716490.6940.3510.7330.34925.2
1.22-1.244.40.59320900.7090.2870.6630.35232.2
1.24-1.2750.58827140.7620.2630.6490.36741.3
1.27-1.295.80.54834590.7860.2290.5990.3753.2
1.29-1.326.70.54843090.820.2160.5930.3865.8
1.32-1.357.90.52955380.8550.1930.5660.38584.9
1.35-1.399.60.50561750.8990.1680.5330.3994.8
1.39-1.4211.90.48763130.940.1450.5090.39696.2
1.42-1.4613.10.44563570.9590.1260.4630.41296.5
1.46-1.5113.60.37263230.9730.1040.3860.43196.8
1.51-1.5713.60.30163790.9820.0830.3130.44797.1
1.57-1.6313.50.25263970.9860.070.2620.46697.3
1.63-1.713.30.21164710.990.0590.2190.48197.6
1.7-1.7912.50.1764420.9910.050.1770.51397.8
1.79-1.912.70.12665020.9960.0360.1310.56898.1
1.9-2.0513.80.09465490.9980.0260.0980.64998.4
2.05-2.26140.07465310.9990.020.0770.73498.6
2.26-2.5913.70.06466580.9990.0180.0660.83298.9
2.59-3.2612.50.05267200.9990.0150.0541.02699.2
3.26-5013.20.04270400.9990.0120.0441.37599.3

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Phasing

PhasingMethod: molecular replacement
Phasing MR
Highest resolutionLowest resolution
Rotation5.62 Å35.59 Å
Translation5.62 Å35.59 Å

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
HKL-2000data reduction
HKL-2000data scaling
PHASER2.8.3phasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.2→35.59 Å / SU ML: 0.0844 / Cross valid method: THROUGHOUT / σ(F): 1.37 / Phase error: 15.6197
RfactorNum. reflection% reflection
Rfree0.16 2000 1.81 %
Rwork0.1375 --
obs0.1379 110584 83.63 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 15.3 Å2
Refinement stepCycle: final / Resolution: 1.2→35.59 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3167 0 36 420 3623
Biso mean--20.94 27.97 -
Num. residues----394
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00493718
X-RAY DIFFRACTIONf_angle_d0.8225126
X-RAY DIFFRACTIONf_chiral_restr0.0832555
X-RAY DIFFRACTIONf_plane_restr0.0038673
X-RAY DIFFRACTIONf_dihedral_angle_d7.86952367
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.2-1.230.3286440.26242417X-RAY DIFFRACTION26.38
1.23-1.260.2976640.21123458X-RAY DIFFRACTION37.71
1.26-1.30.2407910.18334914X-RAY DIFFRACTION53.8
1.3-1.340.23421250.16666852X-RAY DIFFRACTION74.52
1.34-1.390.1881610.15188663X-RAY DIFFRACTION94.52
1.39-1.450.17191620.12658849X-RAY DIFFRACTION96.37
1.45-1.510.1551640.11228916X-RAY DIFFRACTION96.75
1.51-1.590.12361670.10359011X-RAY DIFFRACTION97.18
1.59-1.690.14871640.10828962X-RAY DIFFRACTION97.53
1.69-1.820.14981680.11959090X-RAY DIFFRACTION97.92
1.82-2.010.1441690.11959133X-RAY DIFFRACTION98.27
2.01-2.30.14321700.12939237X-RAY DIFFRACTION98.69
2.3-2.890.1621710.14989337X-RAY DIFFRACTION99.13
2.89-35.590.16161800.15069745X-RAY DIFFRACTION99.52

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