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- EMDB-3031: Cryo-EM structure of ATP-bound IstB in complex to duplex DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-3031
TitleCryo-EM structure of ATP-bound IstB in complex to duplex DNA
Map dataIstB decamer bound to duplex DNA
Sample
  • Sample: ATP-bound IstB decamer bound to a 60 bp duplex DNA
  • Protein or peptide: IstB
  • DNA: Duplex DNA
KeywordsAAA+ / ATPase / IS21 / DNA transposition / DNA binding / DNA remodeling
Biological speciesGeobacillus stearothermophilus (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsArias-Palomo E / Berger JM
CitationJournal: Cell / Year: 2015
Title: An Atypical AAA+ ATPase Assembly Controls Efficient Transposition through DNA Remodeling and Transposase Recruitment.
Authors: Ernesto Arias-Palomo / James M Berger /
Abstract: Transposons are ubiquitous genetic elements that drive genome rearrangements, evolution, and the spread of infectious disease and drug-resistance. Many transposons, such as Mu, Tn7, and IS21, require ...Transposons are ubiquitous genetic elements that drive genome rearrangements, evolution, and the spread of infectious disease and drug-resistance. Many transposons, such as Mu, Tn7, and IS21, require regulatory AAA+ ATPases for function. We use X-ray crystallography and cryo-electron microscopy to show that the ATPase subunit of IS21, IstB, assembles into a clamshell-shaped decamer that sandwiches DNA between two helical pentamers of ATP-associated AAA+ domains, sharply bending the duplex into a 180° U-turn. Biochemical studies corroborate key features of the structure and further show that the IS21 transposase, IstA, recognizes the IstB•DNA complex and promotes its disassembly by stimulating ATP hydrolysis. Collectively, these studies reveal a distinct manner of higher-order assembly and client engagement by a AAA+ ATPase and suggest a mechanistic model where IstB binding and subsequent DNA bending primes a selected insertion site for efficient transposition.
History
DepositionMay 29, 2015-
Header (metadata) releaseJun 10, 2015-
Map releaseAug 26, 2015-
UpdateAug 26, 2015-
Current statusAug 26, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3031.png

Downloads & links

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Map

FileDownload / File: emd_3031.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationIstB decamer bound to duplex DNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.05 Å/pix.
x 144 pix.
= 295.2 Å		2.05 Å/pix.
x 144 pix.
= 295.2 Å		2.05 Å/pix.
x 144 pix.
= 295.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.028
Minimum - Maximum-0.04295128 - 0.10385688
Average (Standard dev.)0.00062353 (±0.00516573)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions144144144
Spacing144144144
CellA=B=C: 295.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.052.052.05
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z295.200295.200295.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS144144144
D min/max/mean-0.0430.1040.001

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Supplemental data

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Sample components

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Entire : ATP-bound IstB decamer bound to a 60 bp duplex DNA

EntireName: ATP-bound IstB decamer bound to a 60 bp duplex DNA
Components
  • Sample: ATP-bound IstB decamer bound to a 60 bp duplex DNA
  • Protein or peptide: IstB
  • DNA: Duplex DNA

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Supramolecule #1000: ATP-bound IstB decamer bound to a 60 bp duplex DNA

SupramoleculeName: ATP-bound IstB decamer bound to a 60 bp duplex DNA / type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: One IstB pentamer of dimers binds to a 50-basepair DNA
Number unique components: 2
Molecular weightExperimental: 320 KDa / Theoretical: 320 KDa / Method: Analytical size exclusion chromatography

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Macromolecule #1: IstB

MacromoleculeName: IstB / type: protein_or_peptide / ID: 1 / Number of copies: 10 / Oligomeric state: Decamer / Recombinant expression: Yes
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Molecular weightTheoretical: 29 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: Duplex DNA

MacromoleculeName: Duplex DNA / type: dna / ID: 2 / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
SequenceString:
CCTGAATTGT TAAAGCCGAT GATAAAATCC CCAATATAGC CGGAATAAAA TTCCCCACTT

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.012 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES pH 7.5, 300 mM NaCl, 5 mM MgCl2, 1.5% glycerol, 1 mM DTT, 1 mM ATP
GridDetails: C-Flat 400 mesh 2/2 with thin carbon support
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK II / Method: Blot for 4 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
DateMay 23, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 100000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each micrograph
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: OTHER / Software - Name: EMAN2, SPARX / Number images used: 263191

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