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- PDB-6vsx: X-ray crystal structure of the C-terminal domain of Bacillus subt... -

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Basic information

Entry
Database: PDB / ID: 6vsx
TitleX-ray crystal structure of the C-terminal domain of Bacillus subtilis RNA polymerase binding helicase HelD
ComponentsDNA helicaseHelicase
KeywordsTRANSCRIPTION / RNA polymerase / Helicase
Function / homology
Function and homology information


recombinational repair / 3'-5' DNA helicase activity / DNA helicase activity / DNA helicase / hydrolase activity / ATP hydrolysis activity / DNA binding / ATP binding / cytosol
Similarity search - Function
: / UvrD-like helicase C-terminal domain / UvrD-like helicase C-terminal domain / UvrD/REP helicase N-terminal domain / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / P-loop containing nucleotide triphosphate hydrolases / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold ...: / UvrD-like helicase C-terminal domain / UvrD-like helicase C-terminal domain / UvrD/REP helicase N-terminal domain / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / P-loop containing nucleotide triphosphate hydrolases / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHATE ION / ATP-dependent DNA helicase rep / DNA helicase IV
Similarity search - Component
Biological speciesBacillus subtilis (bacteria)
MethodX-RAY DIFFRACTION / SAD / Resolution: 2 Å
AuthorsMurakami, K.S. / Chon, U.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131860 United States
CitationJournal: Nat Commun / Year: 2020
Title: Mycobacterial HelD is a nucleic acids-clearing factor for RNA polymerase.
Authors: Tomáš Kouba / Tomáš Koval' / Petra Sudzinová / Jiří Pospíšil / Barbora Brezovská / Jarmila Hnilicová / Hana Šanderová / Martina Janoušková / Michaela Šiková / Petr Halada / ...Authors: Tomáš Kouba / Tomáš Koval' / Petra Sudzinová / Jiří Pospíšil / Barbora Brezovská / Jarmila Hnilicová / Hana Šanderová / Martina Janoušková / Michaela Šiková / Petr Halada / Michal Sýkora / Ivan Barvík / Jiří Nováček / Mária Trundová / Jarmila Dušková / Tereza Skálová / URee Chon / Katsuhiko S Murakami / Jan Dohnálek / Libor Krásný /
Abstract: RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism ...RNA synthesis is central to life, and RNA polymerase (RNAP) depends on accessory factors for recovery from stalled states and adaptation to environmental changes. Here, we investigated the mechanism by which a helicase-like factor HelD recycles RNAP. We report a cryo-EM structure of a complex between the Mycobacterium smegmatis RNAP and HelD. The crescent-shaped HelD simultaneously penetrates deep into two RNAP channels that are responsible for nucleic acids binding and substrate delivery to the active site, thereby locking RNAP in an inactive state. We show that HelD prevents non-specific interactions between RNAP and DNA and dissociates stalled transcription elongation complexes. The liberated RNAP can either stay dormant, sequestered by HelD, or upon HelD release, restart transcription. Our results provide insights into the architecture and regulation of the highly medically-relevant mycobacterial transcription machinery and define HelD as a clearing factor that releases RNAP from nonfunctional complexes with nucleic acids.
History
DepositionFeb 12, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 13, 2021Group: 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.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
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: DNA helicase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)21,3762
Polymers21,2811
Non-polymers951
Water1,964109
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)106.963, 38.813, 44.427
Angle α, β, γ (deg.)90.000, 101.448, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z
Components on special symmetry positions
IDModelComponents
11A-1004-

HOH

21A-1009-

HOH

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Components

#1: Protein DNA helicase / Helicase


Mass: 21281.357 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Gene: B4417_3167 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A164TSE8, UniProt: O32215*PLUS, DNA helicase
#2: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 109 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: 2.12 Å3/Da / Density % sol: 42.08 %
Crystal growTemperature: 300 K / Method: vapor diffusion / pH: 6.2
Details: 0.1M Na/K phosphate, pH 6.2, 0.2 M NaCl, 50 % PEG200

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.5414 Å
DetectorType: RIGAKU SATURN 944+ / Detector: CCD / Date: May 2, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5414 Å / Relative weight: 1
ReflectionResolution: 2→24.73 Å / Num. obs: 11905 / % possible obs: 96.7 % / Redundancy: 20 % / Biso Wilson estimate: 21.97 Å2 / CC1/2: 0.991 / CC star: 0.998 / Rmerge(I) obs: 0.044 / Net I/σ(I): 60.1
Reflection shellResolution: 2→2.03 Å / Rmerge(I) obs: 0.206 / Num. unique obs: 463 / CC1/2: 0.991 / CC star: 0.998 / % possible all: 72

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
HKL-2000data reduction
HKL-2000data scaling
PHENIX1.17.1_3660phasing
PHENIX1.17.1_3660model building
RefinementMethod to determine structure: SAD / Resolution: 2→24.73 Å / SU ML: 0.18 / Cross valid method: FREE R-VALUE / σ(F): 0 / Phase error: 19.615
RfactorNum. reflection% reflection
Rfree0.2015 1174 10.01 %
Rwork0.1723 --
obs0.1752 11730 95.15 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 25.23 Å2
Refinement stepCycle: LAST / Resolution: 2→24.73 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1268 0 5 109 1382
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00721299
X-RAY DIFFRACTIONf_angle_d0.80211755
X-RAY DIFFRACTIONf_chiral_restr0.0538197
X-RAY DIFFRACTIONf_plane_restr0.0048223
X-RAY DIFFRACTIONf_dihedral_angle_d20.061172
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2-2.090.23331180.17231047X-RAY DIFFRACTION76.04
2.09-2.20.22561460.1611304X-RAY DIFFRACTION94.96
2.2-2.340.23081480.16471353X-RAY DIFFRACTION98.17
2.34-2.520.19451480.18181329X-RAY DIFFRACTION97.17
2.52-2.770.1971500.17951356X-RAY DIFFRACTION97.54
2.77-3.170.22721530.18721350X-RAY DIFFRACTION97.28
3.17-3.990.17051520.16181382X-RAY DIFFRACTION100
3.99-24.730.19731590.17271435X-RAY DIFFRACTION99.81

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