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- PDB-7awk: Crystal structure of the HigB1 toxin mutant K95A from Mycobacteri... -

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Basic information

Entry
Database: PDB / ID: 7awk
TitleCrystal structure of the HigB1 toxin mutant K95A from Mycobacterium tuberculosis (Rv1955)
ComponentsProbable endoribonuclease HigB1
KeywordsTOXIN / Mycobacterium tuberculosis / Toxin-Antitoxin-Chaperone / RNase
Function / homologyToxin HigB-like / Phage derived protein Gp49-like (DUF891) / detoxification / negative regulation of growth / RNA endonuclease activity / Hydrolases; Acting on ester bonds / response to hypoxia / Probable endoribonuclease HigB1
Function and homology information
Biological speciesMycobacterium tuberculosis (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.91 Å
AuthorsBigot, D.J. / Guillet, V. / Mourey, L.
Funding support France, 1items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-189-CE12-0026-03 France
CitationJournal: Nat Commun / Year: 2022
Title: Substrate recognition and cryo-EM structure of the ribosome-bound TAC toxin of Mycobacterium tuberculosis.
Authors: Moise Mansour / Emmanuel Giudice / Xibing Xu / Hatice Akarsu / Patricia Bordes / Valérie Guillet / Donna-Joe Bigot / Nawel Slama / Gaetano D'urso / Sophie Chat / Peter Redder / Laurent ...Authors: Moise Mansour / Emmanuel Giudice / Xibing Xu / Hatice Akarsu / Patricia Bordes / Valérie Guillet / Donna-Joe Bigot / Nawel Slama / Gaetano D'urso / Sophie Chat / Peter Redder / Laurent Falquet / Lionel Mourey / Reynald Gillet / Pierre Genevaux /
Abstract: Toxins of toxin-antitoxin systems use diverse mechanisms to control bacterial growth. Here, we focus on the deleterious toxin of the atypical tripartite toxin-antitoxin-chaperone (TAC) system of ...Toxins of toxin-antitoxin systems use diverse mechanisms to control bacterial growth. Here, we focus on the deleterious toxin of the atypical tripartite toxin-antitoxin-chaperone (TAC) system of Mycobacterium tuberculosis, whose inhibition requires the concerted action of the antitoxin and its dedicated SecB-like chaperone. We show that the TAC toxin is a bona fide ribonuclease and identify exact cleavage sites in mRNA targets on a transcriptome-wide scale in vivo. mRNA cleavage by the toxin occurs after the second nucleotide of the ribosomal A-site codon during translation, with a strong preference for CCA codons in vivo. Finally, we report the cryo-EM structure of the ribosome-bound TAC toxin in the presence of native M. tuberculosis cspA mRNA, revealing the specific mechanism by which the TAC toxin interacts with the ribosome and the tRNA in the P-site to cleave its mRNA target.
History
DepositionNov 8, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 24, 2021Provider: repository / Type: Initial release
Revision 1.1May 18, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2May 25, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Probable endoribonuclease HigB1


Theoretical massNumber of molelcules
Total (without water)14,9201
Polymers14,9201
Non-polymers00
Water1,17165
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area6720 Å2
MethodPISA
Unit cell
Length a, b, c (Å)43.975, 82.054, 38.047
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number18
Space group name H-MP21212

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Components

#1: Protein Probable endoribonuclease HigB1 / Toxin HigB1 (Rv1955)


Mass: 14920.277 Da / Num. of mol.: 1 / Mutation: K95A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) (bacteria)
Strain: ATCC 25618 / H37Rv / Gene: higB1, higB, Rv1955 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P9WJA5, Hydrolases; Acting on ester bonds
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 65 / 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: 2.3 Å3/Da / Density % sol: 46.53 %
Crystal growTemperature: 285 K / Method: vapor diffusion, sitting drop
Details: 0.1 M Na acetate pH 4.6, 30 % (w/v) PEG 4K, 0.2 M ammonium acetate
PH range: 4.6-6.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: MASSIF-3 / Wavelength: 0.9677 Å
DetectorType: DECTRIS EIGER X 4M / Detector: PIXEL / Date: Oct 12, 2020
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9677 Å / Relative weight: 1
ReflectionResolution: 1.9→50 Å / Num. obs: 11202 / % possible obs: 98.9 % / Redundancy: 6.7 % / CC1/2: 0.99 / Rrim(I) all: 0.12 / Net I/σ(I): 9.22
Reflection shellResolution: 1.9→1.95 Å / Mean I/σ(I) obs: 1.33 / Num. unique obs: 739 / CC1/2: 0.602 / Rrim(I) all: 1.32 / % possible all: 92.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
XDSdata reduction
XDSdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: Model

Resolution: 1.91→41.027 Å / Cor.coef. Fo:Fc: 0.955 / Cor.coef. Fo:Fc free: 0.923 / SU B: 8.611 / SU ML: 0.127 / Cross valid method: FREE R-VALUE / ESU R: 0.167 / ESU R Free: 0.167
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2734 560 5.004 %
Rwork0.2124 10632 -
all0.215 --
obs-11192 99.299 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 37.644 Å2
Baniso -1Baniso -2Baniso -3
1--0.147 Å20 Å20 Å2
2--0.967 Å2-0 Å2
3----0.82 Å2
Refinement stepCycle: LAST / Resolution: 1.91→41.027 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms973 0 0 65 1038
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0130.0131013
X-RAY DIFFRACTIONr_bond_other_d0.0010.017955
X-RAY DIFFRACTIONr_angle_refined_deg1.8331.651374
X-RAY DIFFRACTIONr_angle_other_deg1.371.5822196
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.3015121
X-RAY DIFFRACTIONr_dihedral_angle_2_deg27.04720.16959
X-RAY DIFFRACTIONr_dihedral_angle_3_deg17.315168
X-RAY DIFFRACTIONr_dihedral_angle_4_deg11.3861510
X-RAY DIFFRACTIONr_chiral_restr0.0870.2127
X-RAY DIFFRACTIONr_gen_planes_refined0.010.021143
X-RAY DIFFRACTIONr_gen_planes_other0.0010.02247
X-RAY DIFFRACTIONr_nbd_refined0.2150.2198
X-RAY DIFFRACTIONr_symmetry_nbd_other0.1850.2866
X-RAY DIFFRACTIONr_nbtor_refined0.1820.2481
X-RAY DIFFRACTIONr_symmetry_nbtor_other0.0890.2459
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1950.252
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_other0.0250.21
X-RAY DIFFRACTIONr_symmetry_nbd_refined0.2310.26
X-RAY DIFFRACTIONr_nbd_other0.2240.232
X-RAY DIFFRACTIONr_symmetry_xyhbond_nbd_refined0.1980.26
X-RAY DIFFRACTIONr_mcbond_it2.4182.815484
X-RAY DIFFRACTIONr_mcbond_other2.422.808483
X-RAY DIFFRACTIONr_mcangle_it3.6434.2605
X-RAY DIFFRACTIONr_mcangle_other3.6434.208606
X-RAY DIFFRACTIONr_scbond_it2.4793.074529
X-RAY DIFFRACTIONr_scbond_other2.4543.059526
X-RAY DIFFRACTIONr_scangle_it3.8554.503769
X-RAY DIFFRACTIONr_scangle_other3.8414.497768
X-RAY DIFFRACTIONr_lrange_it6.40832.4321142
X-RAY DIFFRACTIONr_lrange_other6.32232.1131132
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.91-1.9550.379390.31737X-RAY DIFFRACTION98.2278
1.955-2.0090.266410.287784X-RAY DIFFRACTION100
2.009-2.0670.299370.247706X-RAY DIFFRACTION99.5979
2.067-2.130.269380.224715X-RAY DIFFRACTION99.7351
2.13-2.20.27360.22695X-RAY DIFFRACTION99.8634
2.2-2.2770.224350.204666X-RAY DIFFRACTION99.4326
2.277-2.3630.323340.196638X-RAY DIFFRACTION99.5556
2.363-2.4590.282330.204634X-RAY DIFFRACTION99.701
2.459-2.5680.248320.207604X-RAY DIFFRACTION100
2.568-2.6930.296300.209566X-RAY DIFFRACTION100
2.693-2.8380.242290.252558X-RAY DIFFRACTION100
2.838-3.0090.274280.225530X-RAY DIFFRACTION100
3.009-3.2150.341260.238491X-RAY DIFFRACTION98.8528
3.215-3.4710.239230.247435X-RAY DIFFRACTION92.9006
3.471-3.80.277230.229428X-RAY DIFFRACTION100
3.8-4.2440.197200.186391X-RAY DIFFRACTION100
4.244-4.8910.242190.159352X-RAY DIFFRACTION99.7312
4.891-5.970.314160.186301X-RAY DIFFRACTION99.6855
5.97-8.3540.308120.206247X-RAY DIFFRACTION99.6154
8.354-41.0270.36290.184153X-RAY DIFFRACTION97.006
Refinement TLS params.Method: refined / Origin x: -9.308 Å / Origin y: 11.812 Å / Origin z: -10.992 Å
111213212223313233
T0.009 Å2-0.0083 Å2-0.0084 Å2-0.0323 Å20.0008 Å2--0.1544 Å2
L3.134 °20.8681 °2-1.4168 °2-1.3023 °2-0.7937 °2--2.1232 °2
S0.0591 Å °-0.0566 Å °-0.2374 Å °0.048 Å °-0.1965 Å °-0.0549 Å °0.055 Å °0.0674 Å °0.1373 Å °
Refinement TLS groupSelection: ALL

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