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- PDB-6g1n: Crystal structure of the Burkholderia Pseudomallei antitoxin HicB -

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Basic information

Entry
Database: PDB / ID: 6g1n
TitleCrystal structure of the Burkholderia Pseudomallei antitoxin HicB
Componentsantitoxin HicB
KeywordsANTITOXIN / The antitoxin HicB which acts as an inhibitor to HicA
Function / homologyDouble Stranded RNA Binding Domain - #250 / : / HicB-like antitoxin of toxin-antitoxin system / HicB_like antitoxin of bacterial toxin-antitoxin system / TTHA1013/TTHA0281-like / Double Stranded RNA Binding Domain / 2-Layer Sandwich / Alpha Beta / HicB-like antitoxin of toxin-antitoxin system domain-containing protein
Function and homology information
Biological speciesBurkholderia pseudomallei (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.85 Å
AuthorsWinter, A.J. / Williams, C. / Crump, M.P.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/J014400/1 United Kingdom
CitationJournal: J Biol Chem / Year: 2018
Title: The molecular basis of protein toxin HicA-dependent binding of the protein antitoxin HicB to DNA.
Authors: Ashley J Winter / Christopher Williams / Michail N Isupov / Hannah Crocker / Mariya Gromova / Philip Marsh / Oliver J Wilkinson / Mark S Dillingham / Nicholas J Harmer / Richard W Titball / Matthew P Crump /
Abstract: Toxin-antitoxin (TA) systems are present in many bacteria and play important roles in bacterial growth, physiology, and pathogenicity. Those that are best studied are the type II TA systems, in which ...Toxin-antitoxin (TA) systems are present in many bacteria and play important roles in bacterial growth, physiology, and pathogenicity. Those that are best studied are the type II TA systems, in which both toxins and antitoxins are proteins. The HicAB system is one of the prototypic TA systems, found in many bacterial species. Complex interactions between the protein toxin (HicA), the protein antitoxin (HicB), and the DNA upstream of the encoding genes regulate the activity of this system, but few structural details are available about how HicA destabilizes the HicB-DNA complex. Here, we determined the X-ray structures of HicB and the HicAB complex to 1.8 and 2.5 Å resolution, respectively, and characterized their DNA interactions. This revealed that HicB forms a tetramer and HicA and HicB form a heterooctameric complex that involves structural reorganization of the C-terminal (DNA-binding) region of HicB. Our observations indicated that HicA has a profound impact on binding of HicB to DNA sequences upstream of in a stoichiometric-dependent way. At low ratios of HicA:HicB, there was no effect on DNA binding, but at higher ratios, the affinity for DNA declined cooperatively, driving dissociation of the HicA:HicB:DNA complex. These results reveal the structural mechanisms by which HicA de-represses the HicB-DNA complex.
History
DepositionMar 21, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 31, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 26, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 17, 2024Group: Advisory / Data collection ...Advisory / Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: antitoxin HicB
B: antitoxin HicB
C: antitoxin HicB
D: antitoxin HicB
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,46911
Polymers63,0514
Non-polymers4187
Water3,369187
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Analytical SEC showed a tetrameric association, native gel electrophoresis, Native mass spectrometry showed the presence of a tetramer in the gas phase, SAXS, SAXS showed ...Evidence: gel filtration, Analytical SEC showed a tetrameric association, native gel electrophoresis, Native mass spectrometry showed the presence of a tetramer in the gas phase, SAXS, SAXS showed the formation of a tetramer
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area12550 Å2
ΔGint-105 kcal/mol
Surface area26790 Å2
MethodPISA
Unit cell
Length a, b, c (Å)62.580, 62.580, 173.490
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number76
Space group name H-MP41

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Components

#1: Protein
antitoxin HicB


Mass: 15762.726 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Burkholderia pseudomallei (bacteria) / Strain: K96243 / Gene: BPSS0391 / Production host: Escherichia coli (E. coli) / References: UniProt: Q63NA5
#2: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl
#3: Chemical ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C3H8O3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 187 / Source method: isolated from a natural source / Formula: H2O

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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: 2.67 Å3/Da / Density % sol: 53.6 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop / pH: 4.2 / Details: 0.1 M NaOAc, 0.02M CaCl2.dH20 15% MPD

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Data collection

DiffractionMean temperature: 80 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I03 / Wavelength: 0.9763 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Mar 4, 2017
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9763 Å / Relative weight: 1
ReflectionResolution: 1.85→42.472 Å / Num. obs: 56223 / % possible obs: 99 % / Redundancy: 4.7 % / Biso Wilson estimate: 57.88 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.05854 / Rrim(I) all: 0.066 / Net I/σ(I): 9.84
Reflection shellResolution: 1.85→1.917 Å / Redundancy: 4.7 % / Rmerge(I) obs: 3.521 / Mean I/σ(I) obs: 0.4 / Num. unique obs: 5547 / CC1/2: 0.129 / Rrim(I) all: 3.962 / % possible all: 99

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Processing

Software
NameVersionClassification
PHENIX(1.10.1_2155: ???)refinement
xia2data reduction
DIALSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6G1C

6g1c


Resolution: 1.85→42.472 Å / SU ML: 0.33 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 31.85 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2414 2712 4.86 %
Rwork0.203 --
obs0.2049 55828 98.65 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.85→42.472 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms4120 0 22 187 4329
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0084466
X-RAY DIFFRACTIONf_angle_d0.8676121
X-RAY DIFFRACTIONf_dihedral_angle_d19.2642737
X-RAY DIFFRACTIONf_chiral_restr0.058701
X-RAY DIFFRACTIONf_plane_restr0.006802
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8504-1.88410.46571100.4592513X-RAY DIFFRACTION88
1.8841-1.92030.42571320.42572756X-RAY DIFFRACTION97
1.9203-1.95950.42931360.36222806X-RAY DIFFRACTION99
1.9595-2.00210.381400.33062800X-RAY DIFFRACTION99
2.0021-2.04870.37271430.32804X-RAY DIFFRACTION99
2.0487-2.09990.30861400.27472811X-RAY DIFFRACTION99
2.0999-2.15670.2811680.25772807X-RAY DIFFRACTION99
2.1567-2.22020.29281550.24342758X-RAY DIFFRACTION99
2.2202-2.29180.25811360.23172814X-RAY DIFFRACTION100
2.2918-2.37370.28961760.22672807X-RAY DIFFRACTION100
2.3737-2.46880.27321550.22422791X-RAY DIFFRACTION99
2.4688-2.58110.25821390.21192833X-RAY DIFFRACTION100
2.5811-2.71720.3011520.20912842X-RAY DIFFRACTION100
2.7172-2.88740.26071270.22792803X-RAY DIFFRACTION99
2.8874-3.11020.25981480.21872840X-RAY DIFFRACTION100
3.1102-3.42310.23741370.21352834X-RAY DIFFRACTION100
3.4231-3.91820.251520.18642810X-RAY DIFFRACTION100
3.9182-4.93530.16651220.16262852X-RAY DIFFRACTION99
4.9353-42.48330.20511440.17542835X-RAY DIFFRACTION99
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
13.33110.35651.74653.98551.28227.97840.2482-0.04050.1381-0.0665-0.15910.0937-0.2498-0.8528-0.12320.3537-0.03820.04440.4537-0.00480.396654.748464.815919.8738
22.2120.8744.20633.35063.50068.42030.0745-0.0805-0.12070.3281-0.04370.03030.6578-0.1277-0.00680.4833-0.09530.02850.48170.0190.435157.762455.160520.1482
35.2029-4.0268-0.93144.561.61084.1421-0.4722-0.70150.42270.24770.5125-0.0964-0.02240.0005-0.09720.37840.0812-0.01520.4297-0.0160.429580.792953.316811.8152
42.83490.157-1.36823.3482-2.52967.9333-0.1623-0.046-0.0733-0.04920.1976-0.07150.81820.076-0.0750.4137-0.0450.00840.3331-0.04230.391596.130823.4738-20.0923
52.79340.8096-2.62981.7825-3.91357.9614-0.04930.2832-0.0495-0.27370.1270.20270.3572-0.8947-0.0380.4543-0.1254-0.01030.4515-0.0240.481786.379326.2478-20.4701
64.817-4.1353-1.89475.5211-0.20825.02560.51480.3780.0878-0.8037-0.5074-0.31230.10950.1444-0.02220.45480.06290.00260.4062-0.00730.433784.193449.0722-12.2958
78.9450.1038-2.01219.1172-1.12568.59390.330.4241-0.427-0.0054-0.08491.30850.8629-1.0883-0.1510.4234-0.0725-0.09230.4261-0.07170.368654.334755.39121.2076
84.24741.8633-4.04873.9762-2.64874.0117-0.3486-0.3468-0.7531-0.3468-0.1985-0.33031.07930.61010.44160.54770.0154-0.02640.3712-0.01270.499162.984654.36380.0911
96.26280.9956-5.60090.1023-0.70845.130.40330.17051.44360.36470.38210.2115-0.5826-0.8668-0.57630.55790.12320.07510.55640.02830.607460.568169.656-5.5943
102.1596-1.996-2.49522.71870.14127.10450.2408-0.0484-0.0811-0.0655-0.1828-0.0558-0.5226-0.4067-0.16280.38360.067-0.12920.3736-0.08150.374958.510563.40540.9699
116.64280.32961.163.48020.29184.26320.71951.762-0.1037-2.4161-1.02140.1810.1235-0.2480.28340.91960.4118-0.00170.75690.00090.510882.508652.3002-21.1107
127.15180.12130.80368.26711.3077.9733-0.11890.2593-0.84230.25750.33420.6470.8796-1.0377-0.13060.3949-0.07480.0810.3730.07480.352786.652623.0474-1.4848
133.05262.56812.65214.57955.5218.8183-0.0269-0.3648-0.0602-0.111-0.18120.2739-0.3186-0.93720.11780.3250.03210.01010.39120.0580.424588.233732.32941.6195
143.9137-3.5790.23024.19820.78968.1556-0.046-0.7135-0.21780.56580.4157-0.3541.65280.3287-0.2610.77920.1050.04470.53380.05810.534995.921817.8681-1.4647
159.35355.73642.19956.11623.44688.5501-0.08930.49180.5923-0.24330.1630.5596-0.50150.9512-0.10130.4843-0.01530.04960.4040.07490.472895.978731.7137-4.2012
164.7333-4.8833-2.4195.29343.0943.523-0.2746-0.52391.6706-0.00050.5442-1.2069-0.4850.1001-0.48560.56080.0447-0.03080.4011-0.09270.782791.631452.687311.5835
173.7086-0.77010.08714.6851-0.40952.1348-0.8495-2.4737-0.54291.95240.79940.26210.76870.21640.00590.96970.47990.07451.16190.00760.556582.351149.652622.7137
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1chain 'A' and (resid 1 through 53 )
2X-RAY DIFFRACTION2chain 'A' and (resid 54 through 88 )
3X-RAY DIFFRACTION3chain 'A' and (resid 89 through 131 )
4X-RAY DIFFRACTION4chain 'B' and (resid 1 through 53 )
5X-RAY DIFFRACTION5chain 'B' and (resid 54 through 88 )
6X-RAY DIFFRACTION6chain 'B' and (resid 89 through 132 )
7X-RAY DIFFRACTION7chain 'C' and (resid 1 through 26 )
8X-RAY DIFFRACTION8chain 'C' and (resid 27 through 53 )
9X-RAY DIFFRACTION9chain 'C' and (resid 54 through 65 )
10X-RAY DIFFRACTION10chain 'C' and (resid 66 through 94 )
11X-RAY DIFFRACTION11chain 'C' and (resid 95 through 133 )
12X-RAY DIFFRACTION12chain 'D' and (resid 1 through 26 )
13X-RAY DIFFRACTION13chain 'D' and (resid 27 through 58 )
14X-RAY DIFFRACTION14chain 'D' and (resid 59 through 77 )
15X-RAY DIFFRACTION15chain 'D' and (resid 78 through 88 )
16X-RAY DIFFRACTION16chain 'D' and (resid 89 through 101 )
17X-RAY DIFFRACTION17chain 'D' and (resid 102 through 133 )

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