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- EMDB-5698: Structure of NtrC1 ATPase in complex with Sigma-54 and promoter DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-5698
TitleStructure of NtrC1 ATPase in complex with Sigma-54 and promoter DNA
Map dataNegative stained reconstruction of NtrC1 AAA+ ATPase, Sigma-54, and promoter DNA complex
Sample
  • Sample: Complex of NtrC1 AAA+ ATPase hexamer with Sigma-54 and promoter DNA
  • Protein or peptide: ATPase Associated with various cellular activities
  • Protein or peptide: Sigma-54
  • DNA: nifH promoter DNA
KeywordsAAA+ ATPase / NtrC1 / Sigma-54
Function / homology
Function and homology information


DNA-binding transcription activator activity / sigma factor activity / nucleotidyltransferase activity / DNA-directed RNA polymerase complex / ribonucleoside triphosphate phosphatase activity / DNA-templated transcription initiation / DNA-directed RNA polymerase activity / regulation of DNA-templated transcription / DNA binding
Similarity search - Function
Sigma-54 factors family signature 1. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54 ...Sigma-54 factors family signature 1. / RNA polymerase sigma factor 54, core-binding domain / RNA polymerase sigma factor 54, DNA-binding / RNA polymerase sigma-54 factor, core-binding domain superfamily / Sigma-54 factor, Activator interacting domain (AID) / Sigma-54, DNA binding domain / Sigma-54 factor, core binding domain / Sigma-54 factors family signature 2. / Sigma-54 factors family profile. / RNA polymerase sigma factor 54 / RNA polymerase sigma factor 54 / AAA+ ATPase domain
Similarity search - Domain/homology
RNA polymerase sigma-54 factor
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria) / Klebsiella pneumoniae (bacteria) / Sinorhizobium meliloti (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 24.0 Å
AuthorsChowdhury S / Sysoeva TA / Guo L / Nixon BT
CitationJournal: Genes Dev / Year: 2013
Title: Nucleotide-induced asymmetry within ATPase activator ring drives σ54-RNAP interaction and ATP hydrolysis.
Authors: Tatyana A Sysoeva / Saikat Chowdhury / Liang Guo / B Tracy Nixon /
Abstract: It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, ...It is largely unknown how the typical homomeric ring geometry of ATPases associated with various cellular activities enables them to perform mechanical work. Small-angle solution X-ray scattering, crystallography, and electron microscopy (EM) reconstructions revealed that partial ATP occupancy caused the heptameric closed ring of the bacterial enhancer-binding protein (bEBP) NtrC1 to rearrange into a hexameric split ring of striking asymmetry. The highly conserved and functionally crucial GAFTGA loops responsible for interacting with σ54-RNA polymerase formed a spiral staircase. We propose that splitting of the ensemble directs ATP hydrolysis within the oligomer, and the ring's asymmetry guides interaction between ATPase and the complex of σ54 and promoter DNA. Similarity between the structure of the transcriptional activator NtrC1 and those of distantly related helicases Rho and E1 reveals a general mechanism in homomeric ATPases whereby complex allostery within the ring geometry forms asymmetric functional states that allow these biological motors to exert directional forces on their target macromolecules.
History
DepositionJun 25, 2013-
Header (metadata) releaseNov 20, 2013-
Map releaseNov 20, 2013-
UpdateAug 27, 2014-
Current statusAug 27, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.75
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5698_1.tif

Downloads & links

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Map

FileDownload / File: emd_5698.map.gz / Format: CCP4 / Size: 373 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stained reconstruction of NtrC1 AAA+ ATPase, Sigma-54, and promoter DNA complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.02 Å/pix.
x 46 pix.
= 276.736 Å		6.02 Å/pix.
x 46 pix.
= 276.736 Å		6.02 Å/pix.
x 46 pix.
= 276.736 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 6.016 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.75 / Movie #1: 2.75
Minimum - Maximum-4.88061285 - 15.494498249999999
Average (Standard dev.)0.11384889 (±0.82977331)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-22-22-22
Dimensions464646
Spacing464646
CellA=B=C: 276.736 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.0166.0166.016
M x/y/z464646
origin x/y/z0.0000.0000.000
length x/y/z276.736276.736276.736
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-22-22-22
NC/NR/NS464646
D min/max/mean-4.88115.4940.114

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Supplemental data

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Sample components

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Entire : Complex of NtrC1 AAA+ ATPase hexamer with Sigma-54 and promoter DNA

EntireName: Complex of NtrC1 AAA+ ATPase hexamer with Sigma-54 and promoter DNA
Components
  • Sample: Complex of NtrC1 AAA+ ATPase hexamer with Sigma-54 and promoter DNA
  • Protein or peptide: ATPase Associated with various cellular activities
  • Protein or peptide: Sigma-54
  • DNA: nifH promoter DNA

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Supramolecule #1000: Complex of NtrC1 AAA+ ATPase hexamer with Sigma-54 and promoter DNA

SupramoleculeName: Complex of NtrC1 AAA+ ATPase hexamer with Sigma-54 and promoter DNA
type: sample / ID: 1000 / Details: The sample was monodisperse.
Oligomeric state: One hexamer of NtrC1 AAA+ ATPase binds to one double-stranded promoter DNA and one Sigma-54.
Number unique components: 3
Molecular weightTheoretical: 260 KDa

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Macromolecule #1: ATPase Associated with various cellular activities

MacromoleculeName: ATPase Associated with various cellular activities / type: protein_or_peptide / ID: 1 / Name.synonym: AAA+ ATPase, Sigma-54 transcription activator / Details: Hexamer of the NtrC1 AAA+ ATPase domain / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Aquifex aeolicus (bacteria) / Strain: VF5
Molecular weightTheoretical: 31 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET122
SequenceGO: ribonucleoside triphosphate phosphatase activity / InterPro: AAA+ ATPase domain

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Macromolecule #2: Sigma-54

MacromoleculeName: Sigma-54 / type: protein_or_peptide / ID: 2
Name.synonym: Sigma-54 component of bacterial RNA polymerase
Details: Single copy of bacterial transcription initiation factor Sigma-54
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Molecular weightTheoretical: 57 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(DE3) / Recombinant plasmid: pET122
SequenceUniProtKB: RNA polymerase sigma-54 factor
GO: DNA-templated transcription initiation, regulation of DNA-templated transcription, DNA binding, DNA-directed RNA polymerase activity, sigma factor activity
InterPro: RNA polymerase sigma factor 54

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Macromolecule #3: nifH promoter DNA

MacromoleculeName: nifH promoter DNA / type: dna / ID: 3 / Name.synonym: Sigma-54 promoter DNA
Details: This is a pre-melt nifH promoter, with a -11, -12 mismatch. The complementary strand has G and T at 12 and 13 base positions from 5' end instead of T and G.
Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Sinorhizobium meliloti (bacteria)
Molecular weightTheoretical: 22 KDa
SequenceString:
CAGACGGCTG GCACGACTTT TGCCAGATCA GCCCTG

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.9
Details: 1mM ADP, 1mM AlCl3, 8mM NaF, 1mM MgCl2, 20mM Tris-HCl, 1% (w/v) trehalose, 1mM TCEP
StainingType: NEGATIVE
Details: Sample was adsorbed on thin continuous carbon coated grids, stained with 0.75% (w/v) uranyl formate, and air-dried.
GridDetails: Thin carbon film on 300 mesh Cu-Rh maxtaform grids, plasma cleaned in oxygen-hydrogen gas mixture for 15s
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2100F
DateAug 10, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (2k x 2k) / Number real images: 280 / Average electron dose: 20 e/Å2 / Camera length: 49
Details: 102 regular, untilted micrographs were collected and 89 tilt-untilt pairs of RCT micrographs were collected.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Room temperature specimen / Specimen holder model: JEOL / Tilt angle min: 45 / Tilt angle max: 65

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Image processing

DetailsParticles were picked manually using XMIPP.
CTF correctionDetails: Per micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: XMIPP, EMAN2, SPARX
Details: Final map was calculated by refinement of RCT model by iterative projection matching and back projection.
Number images used: 20000
Final angle assignmentDetails: EMAN2: az 90 degrees, alt 90 degrees, phi 90 degrees

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