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- EMDB-1409: The EM structure of human DNA polymerase gamma reveals a localize... -

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Basic information

Entry
Database: EMDB / ID: EMD-1409
TitleThe EM structure of human DNA polymerase gamma reveals a localized contact between the catalytic and accessory subunits.
Map dataThis is an image of negatively stained catalytic (A) subunit of human mitochondrial DNA polymerase gamma.
Sample
  • Sample: Catalytic subunit of the human mitochondrial polymerase gamma
  • Protein or peptide: human mitochondrial DNA polymerase gamma, catalytic subunit
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 17.0 Å
AuthorsYakubovskaya E / Lukin M / Chen Z / Berriman J / Wall JS / Kobayashi R / Kisker C / Bogenhagen DF
CitationJournal: EMBO J / Year: 2007
Title: The EM structure of human DNA polymerase gamma reveals a localized contact between the catalytic and accessory subunits.
Authors: Elena Yakubovskaya / Mark Lukin / Zhixin Chen / John Berriman / Joseph S Wall / Ryuji Kobayashi / Caroline Kisker / Daniel F Bogenhagen /
Abstract: We used electron microscopy to examine the structure of human DNA pol gamma, the heterotrimeric mtDNA replicase implicated in certain mitochondrial diseases and aging models. Separate analysis of ...We used electron microscopy to examine the structure of human DNA pol gamma, the heterotrimeric mtDNA replicase implicated in certain mitochondrial diseases and aging models. Separate analysis of negatively stained preparations of the catalytic subunit, pol gammaA, and of the holoenzyme including a dimeric accessory factor, pol gammaB(2), permitted unambiguous identification of the position of the accessory factor within the holoenzyme. The model explains protection of a partial chymotryptic cleavage site after residue L(549) of pol gammaA upon binding of the accessory subunit. This interaction region is near residue 467 of pol gammaA, where a disease-related mutation has been reported to impair binding of the B subunit. One pol gammaB subunit dominates contacts with the catalytic subunit, while the second B subunit is largely exposed to solvent. A model for pol gamma is discussed that considers the effects of known mutations in the accessory subunit and the interaction of the enzyme with DNA.
History
DepositionAug 7, 2007-
Header (metadata) releaseAug 7, 2007-
Map releaseJan 7, 2008-
UpdateMar 26, 2014-
Current statusMar 26, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.839065776
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.839065776
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1409.gif

Downloads & links

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Map

FileDownload / File: emd_1409.map.gz / Format: CCP4 / Size: 422.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an image of negatively stained catalytic (A) subunit of human mitochondrial DNA polymerase gamma.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.49 Å/pix.
x 48 pix.
= 167.424 Å		3.49 Å/pix.
x 48 pix.
= 167.424 Å		3.49 Å/pix.
x 48 pix.
= 167.424 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.488 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.626 / Movie #1: 0.8390658
Minimum - Maximum-0.519939 - 2.60958
Average (Standard dev.)-0.0000656148 (±0.302771)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-24-24-24
Dimensions484848
Spacing484848
CellA=B=C: 167.424 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.4883.4883.488
M x/y/z484848
origin x/y/z0.0000.0000.000
length x/y/z167.424167.424167.424
α/β/γ90.00090.00090.000
start NX/NY/NZ-50-50-50
NX/NY/NZ100100100
MAP C/R/S123
start NC/NR/NS-24-24-24
NC/NR/NS484848
D min/max/mean-0.5202.610-0.000

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Supplemental data

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Sample components

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Entire : Catalytic subunit of the human mitochondrial polymerase gamma

EntireName: Catalytic subunit of the human mitochondrial polymerase gamma
Components
  • Sample: Catalytic subunit of the human mitochondrial polymerase gamma
  • Protein or peptide: human mitochondrial DNA polymerase gamma, catalytic subunit

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Supramolecule #1000: Catalytic subunit of the human mitochondrial polymerase gamma

SupramoleculeName: Catalytic subunit of the human mitochondrial polymerase gamma
type: sample / ID: 1000 / Number unique components: 1
Molecular weightExperimental: 145 KDa / Theoretical: 145 KDa

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Macromolecule #1: human mitochondrial DNA polymerase gamma, catalytic subunit

MacromoleculeName: human mitochondrial DNA polymerase gamma, catalytic subunit
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Location in cell: mitochondria
Molecular weightExperimental: 145 KDa / Theoretical: 145 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: SF9

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.025 mg/mL
BufferpH: 8
Details: 20 mM Hepes, 150 mM KCl, 1 mM EDTA, 5 mM DTT buffer.
StainingType: NEGATIVE
Details: Grids with adsorbed protein were stained with 2% uranyl acetate for 60 seconds.
GridDetails: 300-mesh copper grids (Ted Pella) coated with carbon film that were glow-discharged for 10 s.
VitrificationCryogen name: ETHANE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM200T
Specialist opticsEnergy filter - Name: FEI
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Number real images: 24 / Average electron dose: 10 e/Å2
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 76000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.6 mm / Nominal defocus max: 1.9 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN HELIUM / Tilt angle min: 60

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 8764
Final two d classificationNumber classes: 25

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