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- EMDB-2395: MuB is an AAA+ ATPase that forms helical filaments to control tar... -

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Basic information

Entry
Database: EMDB / ID: EMD-2395
TitleMuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition
Map dataReconstruction of MuB filament without DNA
Sample
  • Sample: MuB filament without DNA
  • Protein or peptide: MuB AAA+ ATPase
KeywordsAAA+ ATPase / DNA transposition / Mu phage / nucleoprotein filament / symmetry mismatch
Function / homology
Function and homology information


DNA-binding transcription activator activity / phosphorelay signal transduction system / cis-regulatory region sequence-specific DNA binding / protein-DNA complex / positive regulation of DNA-templated transcription / ATP hydrolysis activity / ATP binding / metal ion binding / identical protein binding
Similarity search - Function
Sigma-54 interaction domain ATP-binding region A signature. / Sigma-54 interaction domain, ATP-binding site 1 / Sigma-54 interaction domain, ATP-binding site 2 / Sigma-54 interaction domain ATP-binding region B signature. / Sigma-54 interaction domain profile. / Sigma-54 interaction domain / RNA polymerase sigma factor 54 interaction domain / DNA binding HTH domain, Fis-type / Bacterial regulatory protein, Fis family / Response regulator receiver domain ...Sigma-54 interaction domain ATP-binding region A signature. / Sigma-54 interaction domain, ATP-binding site 1 / Sigma-54 interaction domain, ATP-binding site 2 / Sigma-54 interaction domain ATP-binding region B signature. / Sigma-54 interaction domain profile. / Sigma-54 interaction domain / RNA polymerase sigma factor 54 interaction domain / DNA binding HTH domain, Fis-type / Bacterial regulatory protein, Fis family / Response regulator receiver domain / cheY-homologous receiver domain / Signal transduction response regulator, receiver domain / Response regulatory domain profile. / CheY-like superfamily / Homeobox-like domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Transcriptional regulator (NtrC family)
Similarity search - Component
Biological speciesEnterobacteria phage Mu (virus)
Methodhelical reconstruction / cryo EM / Resolution: 18.0 Å
AuthorsMizuno N / Dramicanin M / Mizuuchi M / Adam J / Wang Y / Han YW / Yang W / Steven AC / Mizuuchi K / Ramon-Maiques S
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: MuB is an AAA+ ATPase that forms helical filaments to control target selection for DNA transposition.
Authors: Naoko Mizuno / Marija Dramićanin / Michiyo Mizuuchi / Julia Adam / Yi Wang / Yong-Woon Han / Wei Yang / Alasdair C Steven / Kiyoshi Mizuuchi / Santiago Ramón-Maiques /
Abstract: MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has ...MuB is an ATP-dependent nonspecific DNA-binding protein that regulates the activity of the MuA transposase and captures target DNA for transposition. Mechanistic understanding of MuB function has previously been hindered by MuB's poor solubility. Here we combine bioinformatic, mutagenic, biochemical, and electron microscopic analyses to unmask the structure and function of MuB. We demonstrate that MuB is an ATPase associated with diverse cellular activities (AAA+ ATPase) and forms ATP-dependent filaments with or without DNA. We also identify critical residues for MuB's ATPase, DNA binding, protein polymerization, and MuA interaction activities. Using single-particle electron microscopy, we show that MuB assembles into a helical filament, which binds the DNA in the axial channel. The helical parameters of the MuB filament do not match those of the coated DNA. Despite this protein-DNA symmetry mismatch, MuB does not deform the DNA duplex. These findings, together with the influence of MuB filament size on strand-transfer efficiency, lead to a model in which MuB-imposed symmetry transiently deforms the DNA at the boundary of the MuB filament and results in a bent DNA favored by MuA for transposition.
History
DepositionJun 5, 2013-
Header (metadata) releaseJul 3, 2013-
Map releaseJul 3, 2013-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4bs1
  • Surface level: 0.019
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4bs1
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2395.png

Downloads & links

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Map

FileDownload / File: emd_2395.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of MuB filament without DNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 80 pix.
= 224. Å		2.8 Å/pix.
x 80 pix.
= 224. Å		2.8 Å/pix.
x 80 pix.
= 224. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.04090111 - 0.06237547
Average (Standard dev.)0.00135403 (±0.01104943)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 224.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z224.000224.000224.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-0.0410.0620.001

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Supplemental data

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Sample components

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Entire : MuB filament without DNA

EntireName: MuB filament without DNA
Components
  • Sample: MuB filament without DNA
  • Protein or peptide: MuB AAA+ ATPase

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Supramolecule #1000: MuB filament without DNA

SupramoleculeName: MuB filament without DNA / type: sample / ID: 1000 / Oligomeric state: helical filament / Number unique components: 1
Molecular weightExperimental: 35 KDa / Theoretical: 35 KDa

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Macromolecule #1: MuB AAA+ ATPase

MacromoleculeName: MuB AAA+ ATPase / type: protein_or_peptide / ID: 1 / Oligomeric state: helical assembly / Recombinant expression: Yes
Source (natural)Organism: Enterobacteria phage Mu (virus)
Molecular weightExperimental: 35 KDa / Theoretical: 35 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.07 mg/mL
BufferpH: 8
Details: 30 mM TrisHCl pH 8.0, 0.15 M KCl, 5mM MgCl2, 1mM DTT, 1 mM ATP or ATP-gamma-S
GridDetails: 300 mesh quantifoil R2/2
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK II / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureMin: 80 K / Max: 85 K / Average: 82 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100,000 times magnification
DateApr 21, 2008
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 1000 (2k x 2k) / Number real images: 129 / Average electron dose: 15 e/Å2
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 38000
Sample stageSpecimen holder: side entry / Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsIHRSR
Final reconstructionApplied symmetry - Helical parameters - Δz: 9.01 Å
Applied symmetry - Helical parameters - Δ&Phi: 66.2 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: spider, bsoft, eman
CTF correctionDetails: phase-flipping

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Atomic model buiding 1

Initial modelPDB ID:

1ny6


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: G
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4bs1:
MuB is an AAAplus ATPase that forms helical filaments to control target selection for DNA transposition

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