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- EMDB-2865: Architecture of the yeast pontin-reptin complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2865
TitleArchitecture of the yeast pontin-reptin complex
Map data3D reconstruction of the yeast pontin-reptin complex
Sample
  • Sample: Complex between the proteins pontin and reptin
  • Protein or peptide: RuvB-like 1
  • Protein or peptide: RuvB-like 2
KeywordsRvb1 / Rvb2 / Tip48 / Tip49 / pontin / reptin / Ino80 / AAA+
Function / homologyTIP49, P-loop domain / Ino80 complex
Function and homology information
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.6 Å
AuthorsTorreira E / Jha S / Lopez-Blanco JR / Arias-Palomo E / Chacon P / Canas C / Ayora S / Dutta A / Llorca O
CitationJournal: Structure / Year: 2008
Title: Architecture of the pontin/reptin complex, essential in the assembly of several macromolecular complexes.
Authors: Eva Torreira / Sudhakar Jha / José R López-Blanco / Ernesto Arias-Palomo / Pablo Chacón / Cristina Cañas / Sylvia Ayora / Anindya Dutta / Oscar Llorca /
Abstract: Pontin and reptin belong to the AAA+ family, and they are essential for the structural integrity and catalytic activity of several chromatin remodeling complexes. They are also indispensable for the ...Pontin and reptin belong to the AAA+ family, and they are essential for the structural integrity and catalytic activity of several chromatin remodeling complexes. They are also indispensable for the assembly of several ribonucleoprotein complexes, including telomerase. Here, we propose a structural model of the yeast pontin/reptin complex based on a cryo-electron microscopy reconstruction at 13 A. Pontin/reptin hetero-dodecamers were purified from in vivo assembled complexes forming a double ring. Two rings interact through flexible domains projecting from each hexamer, constituting an atypical asymmetric form of oligomerization. These flexible domains and the AAA+ cores reveal significant conformational changes when compared with the crystal structure of human pontin that generate enlarged channels. This structure of endogenously assembled pontin/reptin complexes is different than previously described structures, suggesting that pontin and reptin could acquire distinct structural states to regulate their broad functions as molecular motors and scaffolds for nucleic acids and proteins.
History
DepositionAug 25, 2008-
Header (metadata) releaseAug 26, 2008-
Map releaseSep 3, 2010-
UpdateFeb 18, 2011-
Current statusFeb 18, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.78
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.78
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd-2865.gif

Downloads & links

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Map

FileDownload / File: emd_2865.map.gz / Format: CCP4 / Size: 4.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of the yeast pontin-reptin complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.12 Å/pix.
x 104 pix.
= 220.48 Å		2.12 Å/pix.
x 104 pix.
= 220.48 Å		2.12 Å/pix.
x 104 pix.
= 220.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.12 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.786 / Movie #1: 0.78
Minimum - Maximum-0.221553 - 2.02531
Average (Standard dev.)0.0797943 (±0.264807)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions104104104
Spacing104104104
CellA=B=C: 220.48 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z104104104
origin x/y/z0.0000.0000.000
length x/y/z220.480220.480220.480
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS104104104
D min/max/mean-0.2222.0250.080

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Supplemental data

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Sample components

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Entire : Complex between the proteins pontin and reptin

EntireName: Complex between the proteins pontin and reptin
Components
  • Sample: Complex between the proteins pontin and reptin
  • Protein or peptide: RuvB-like 1
  • Protein or peptide: RuvB-like 2

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Supramolecule #1000: Complex between the proteins pontin and reptin

SupramoleculeName: Complex between the proteins pontin and reptin / type: sample / ID: 1000
Oligomeric state: Dodecamer composed of 6 subunits of pontin and 6 subunits of reptin
Number unique components: 2
Molecular weightTheoretical: 650 KDa

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Macromolecule #1: RuvB-like 1

MacromoleculeName: RuvB-like 1 / type: protein_or_peptide / ID: 1 / Name.synonym: Pontin / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 540 KDa
Recombinant expressionOrganism: Insect cells using recombinant baculovirus
SequenceGO: Ino80 complex / InterPro: TIP49, P-loop domain

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Macromolecule #2: RuvB-like 2

MacromoleculeName: RuvB-like 2 / type: protein_or_peptide / ID: 2 / Name.synonym: Reptin / Number of copies: 6 / Oligomeric state: Hexamer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightTheoretical: 540 KDa
Recombinant expressionOrganism: Insect cells using recombinant baculovirus
SequenceGO: Ino80 complex / InterPro: TIP49, P-loop domain

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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

BufferpH: 8 / Details: 25mM TrisHCl, 125mM NaCl
GridDetails: Quantifoil R 2/2 holy grids coated with a thin layer of carbon
VitrificationCryogen name: ETHANE / Chamber temperature: 90 K / Instrument: GATAN CRYOPLUNGE 3 / Details: Vitrification instrument: Gatan Plunger / Method: Blot for a few seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 90 K
Alignment procedureLegacy - Astigmatism: Correction using the astigmator, images collected with the CCD camera and the Digital Micrograph software
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 2.12 µm
Details: Images were scanned at 16 bit-pixel and transformed to 8 bit-pixel prior to particle selection
Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.3 mm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Details9316 particles were extraced in total that revealed significant heterogeneity. These particles were split in three subgroups using unbiased classification procedures in 3D. The final reconstruction was performed using one homogeneous class of particles containing 34.5% of the initial dataset
CTF correctionDetails: Micrograph corrected and only flipping phases
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 3214

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Atomic model buiding 1

Initial modelPDB ID:

2c9o

SoftwareName: ADP_EM
DetailsProtocol: Rigid Body and refinement using a Powell base minimization. Rigid-body fitting was performed using ADP_EM. From the best scores found, we carried out an additional refinement step by freely moving a single monomer using a multidimensional Powel optimization routine. Subsequently, 6-fold rotational symmetry was applied to the refined monomer to complete a hexameric ring but discarding those solutions producing steric clashes with contiguous monomers.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation

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