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- EMDB-1317: Dodecameric structure and ATPase activity of the human TIP48/TIP4... -

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Basic information

Entry
Database: EMDB / ID: EMD-1317
TitleDodecameric structure and ATPase activity of the human TIP48/TIP49 complex.
Map dataThis is the masked 3D map of the TIP48/TIP49 complex
Sample
  • Sample: human TIP48-His6_ TIP49
  • Protein or peptide: TIP48-His6
  • Protein or peptide: TIP49
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 20.0 Å
AuthorsPURI T / WENDLER P / SIGALA B / SAIBIL H / TSANEVA IR
CitationJournal: J Mol Biol / Year: 2007
Title: Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex.
Authors: Teena Puri / Petra Wendler / Barbara Sigala / Helen Saibil / Irina R Tsaneva /
Abstract: TIP48 and TIP49 are two related and highly conserved eukaryotic AAA(+) proteins with an essential biological function and a critical role in major pathways that are closely linked to cancer. They are ...TIP48 and TIP49 are two related and highly conserved eukaryotic AAA(+) proteins with an essential biological function and a critical role in major pathways that are closely linked to cancer. They are found together as components of several highly conserved chromatin-modifying complexes. Both proteins show sequence homology to bacterial RuvB but the nature and mechanism of their biochemical role remain unknown. Recombinant human TIP48 and TIP49 were assembled into a stable high molecular mass equimolar complex and tested for activity in vitro. TIP48/TIP49 complex formation resulted in synergistic increase in ATPase activity but ATP hydrolysis was not stimulated in the presence of single-stranded, double-stranded or four-way junction DNA and no DNA helicase or branch migration activity could be detected. Complexes with catalytic defects in either TIP48 or TIP49 had no ATPase activity showing that both proteins within the TIP48/TIP49 complex are required for ATP hydrolysis. The structure of the TIP48/TIP49 complex was examined by negative stain electron microscopy. Three-dimensional reconstruction at 20 A resolution revealed that the TIP48/TIP49 complex consisted of two stacked hexameric rings with C6 symmetry. The top and bottom rings showed substantial structural differences. Interestingly, TIP48 formed oligomers in the presence of adenine nucleotides, whilst TIP49 did not. The results point to biochemical differences between TIP48 and TIP49, which may explain the structural differences between the two hexameric rings and could be significant for specialised functions that the proteins perform individually.
History
DepositionJan 29, 2007-
Header (metadata) releaseJan 30, 2007-
Map releaseJan 30, 2007-
UpdateMay 26, 2011-
Current statusMay 26, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.252
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.252
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1317.gif

Downloads & links

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Map

FileDownload / File: emd_1317.map.gz / Format: CCP4 / Size: 10.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the masked 3D map of the TIP48/TIP49 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.3 Å/pix.
x 140 pix.
= 462. Å		3.3 Å/pix.
x 140 pix.
= 462. Å		3.3 Å/pix.
x 140 pix.
= 462. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.3 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.305 / Movie #1: 0.252
Minimum - Maximum-4.24058 - 9.95561
Average (Standard dev.)0.0173395 (±0.287573)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 462 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.33.33.3
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z462.000462.000462.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-150-150-149
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-4.2419.9560.017

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Supplemental data

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Sample components

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Entire : human TIP48-His6_ TIP49

EntireName: human TIP48-His6_ TIP49
Components
  • Sample: human TIP48-His6_ TIP49
  • Protein or peptide: TIP48-His6
  • Protein or peptide: TIP49

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Supramolecule #1000: human TIP48-His6_ TIP49

SupramoleculeName: human TIP48-His6_ TIP49 / type: sample / ID: 1000 / Oligomeric state: double hexamer / Number unique components: 2
Molecular weightExperimental: 670 KDa / Theoretical: 609 KDa / Method: size exclusion chromatography

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Macromolecule #1: TIP48-His6

MacromoleculeName: TIP48-His6 / type: protein_or_peptide / ID: 1 / Name.synonym: RUVBL2, 48 kDa TATA box-binding / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / Strain: IMAGE2819778 / synonym: human
Molecular weightExperimental: 52 KDa
Recombinant expressionOrganism: Escherichia coli BL21 Gold DE3 / Recombinant plasmid: pET21

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Macromolecule #2: TIP49

MacromoleculeName: TIP49 / type: protein_or_peptide / ID: 2 / Name.synonym: RUVBL1, 49 kDa TATA box-binding / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / Strain: IMAGE2823568 / synonym: human
Molecular weightExperimental: 51.2 KDa / Theoretical: 55 KDa
Recombinant expressionOrganism: Escherichia coli BL21 Gold DE3 / Recombinant plasmid: pET21

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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

Concentration0.063 mg/mL
BufferpH: 8
Details: 20mM Tris HCL pH 8.0, 100 mM NaCl, 5%(w/v)glycerol, 1mM DTT
StainingType: NEGATIVE
Details: grids with adsorbed protein were stained twice with 2% (w/v) uranyl acetate
GridDetails: carbon coated copper grids, 400 mesh, negatively glow discharged
VitrificationCryogen name: NONE

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 1.014 µm / Nominal defocus min: 0.383 µm / Nominal magnification: 42000
Sample stageSpecimen holder: side entry / Specimen holder model: OTHER
TemperatureAverage: 273 K
Alignment procedureLegacy - Astigmatism: corrected at 150,000 magnification
DateSep 3, 2004
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 9 / Average electron dose: 20 e/Å2 / Od range: 1 / Bits/pixel: 8

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Image processing

CTF correctionDetails: phase flipping
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Imagic, Spider / Number images used: 1765

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