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- EMDB-10996: Structure of Mycobacterium smegmatis HelD protein in complex with... -

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Basic information

Entry
Database: EMDB / ID: EMD-10996
TitleStructure of Mycobacterium smegmatis HelD protein in complex with RNA polymerase core - State I, primary channel engaged
Map data
SampleMycobacterium smegmatis HelD protein in complex with RNA polymerase core
  • (DNA-directed RNA polymerase subunit ...Polymerase) x 4
  • RNA polymerase-associated transcription factor HelD
  • (ligand) x 2
Function / homology
Function and homology information


ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA helicase / DNA helicase activity / transcription, DNA-templated / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding ...ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / DNA helicase / DNA helicase activity / transcription, DNA-templated / protein dimerization activity / response to antibiotic / magnesium ion binding / DNA binding / zinc ion binding / ATP binding
RNA polymerase Rpb2, domain 3 / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion / P-loop containing nucleoside triphosphate hydrolase / RNA polymerase, alpha subunit, C-terminal / RNA polymerase Rpb1, domain 5 ...RNA polymerase Rpb2, domain 3 / DNA-directed RNA polymerase beta subunit, bacterial-type / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, protrusion / P-loop containing nucleoside triphosphate hydrolase / RNA polymerase, alpha subunit, C-terminal / RNA polymerase Rpb1, domain 5 / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / DNA-directed RNA polymerase, alpha subunit / RNA polymerase subunit, RPB6/omega / DNA-directed RNA polymerase, subunit beta-prime / UvrD-like helicase, ATP-binding domain / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 1 / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / DNA-directed RNA polymerase, insert domain superfamily / DNA-directed RNA polymerase, subunit 2 / RNA polymerase, RBP11-like subunit / RPB6/omega subunit-like superfamily / DNA helicase, UvrD/REP type / RNA polymerase, alpha subunit / DNA-directed RNA polymerase, omega subunit / RNA polymerase, subunit omega/K/RPB6 / RNA polymerase, N-terminal
DNA-directed RNA polymerase subunit beta' / DNA-directed RNA polymerase subunit alpha / DNA helicase / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.08 Å
AuthorsKouba T / Koval T / Krasny L / Dohnalek J
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.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMay 3, 2020-
Header (metadata) releaseNov 4, 2020-
Map releaseNov 4, 2020-
UpdateNov 4, 2020-
Current statusNov 4, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.17
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.17
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6yxu
  • Surface level: 0.17
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10996.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 299.196 Å
0.83 Å/pix.
x 360 pix.
= 299.196 Å
0.83 Å/pix.
x 360 pix.
= 299.196 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8311 Å
Density
Contour LevelBy AUTHOR: 0.17 / Movie #1: 0.17
Minimum - Maximum-0.21540773 - 0.69156736
Average (Standard dev.)0.0026655702 (±0.02550039)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 299.19598 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.83110.83110.8311
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z299.196299.196299.196
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.2150.6920.003

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Supplemental data

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Additional map: Structure of Mycobacterium smegmatis RNA polymerase core in...

Fileemd_10996_additional_1.map
AnnotationStructure of Mycobacterium smegmatis RNA polymerase core in complex with HelD protein (State I)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Structure of Mycobacterium smegmatis RNA polymerase core in...

Fileemd_10996_half_map_1.map
AnnotationStructure of Mycobacterium smegmatis RNA polymerase core in complex with HelD protein (State I) halfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Structure of Mycobacterium smegmatis RNA polymerase core in...

Fileemd_10996_half_map_2.map
AnnotationStructure of Mycobacterium smegmatis RNA polymerase core in complex with HelD protein (State I) halfmap 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Mycobacterium smegmatis HelD protein in complex with RNA polymera...

EntireName: Mycobacterium smegmatis HelD protein in complex with RNA polymerase core
Number of components: 8

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Component #1: protein, Mycobacterium smegmatis HelD protein in complex with RNA...

ProteinName: Mycobacterium smegmatis HelD protein in complex with RNA polymerase core
Recombinant expression: No
MassTheoretical: 444 kDa
SourceSpecies: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #2: protein, DNA-directed RNA polymerase subunit alpha

ProteinName: DNA-directed RNA polymerase subunit alphaPolymerase / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 37.959441 kDa
SourceSpecies: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, DNA-directed RNA polymerase subunit beta

ProteinName: DNA-directed RNA polymerase subunit betaPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 128.680141 kDa
SourceSpecies: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, DNA-directed RNA polymerase subunit beta'

ProteinName: DNA-directed RNA polymerase subunit beta'Polymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 146.712891 kDa
SourceSpecies: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #5: protein, DNA-directed RNA polymerase subunit omega

ProteinName: DNA-directed RNA polymerase subunit omegaPolymerase / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.544763 kDa
SourceSpecies: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #6: protein, RNA polymerase-associated transcription factor HelD

ProteinName: RNA polymerase-associated transcription factor HelD / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 81.298078 kDa
SourceSpecies: Mycolicibacterium smegmatis MC2 155 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #8: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 7.8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 185400
3D reconstructionSoftware: RELION / Resolution: 3.08 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Input PDB model: 6F6W, 6VSX
Output model

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