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- EMDB-6292: An EM structure of the helicase-loader complex in G. kaustophilus... -

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Basic information

Entry
Database: EMDB / ID: EMD-6292
TitleAn EM structure of the helicase-loader complex in G. kaustophilus suggesting an early stage conformation in Gram-positive bacteria
Map dataReconstruction of the GkDnaC-GkDnaI helicase-loader complex in a putative early stage conformation
Sample
  • Sample: GkDnaC-GkDnaI helicase-loader complex
  • Protein or peptide: GkDnaC-GkDnaI complex
KeywordsDNA replication / primosome assembly / helicase-loader complex
Biological speciesGeobacillus kaustophilus (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 19.0 Å
AuthorsLin Y-C / Vankadari N / Hsiao C-D
CitationJournal: To Be Published
Title: An EM structure of the helicase-loader complex in G. kaustophilus suggesting an early stage conformation
Authors: Lin Y-C / Vankadari N / Hsiao C-D
History
DepositionMar 9, 2015-
Header (metadata) releaseApr 22, 2015-
Map releaseMar 23, 2016-
UpdateMar 23, 2016-
Current statusMar 23, 2016Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.066
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.066
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6292.png

Downloads & links

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Map

FileDownload / File: emd_6292.map.gz / Format: CCP4 / Size: 6.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the GkDnaC-GkDnaI helicase-loader complex in a putative early stage conformation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.75 Å/pix.
x 120 pix.
= 210. Å		1.75 Å/pix.
x 120 pix.
= 210. Å		1.75 Å/pix.
x 120 pix.
= 210. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.75 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.066 / Movie #1: 0.066
Minimum - Maximum-0.02440098 - 0.15767528
Average (Standard dev.)0.01170473 (±0.02987073)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions120120120
Spacing120120120
CellA=B=C: 210.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.751.751.75
M x/y/z120120120
origin x/y/z0.0000.0000.000
length x/y/z210.000210.000210.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS120120120
D min/max/mean-0.0240.1580.012

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Supplemental data

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Sample components

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Entire : GkDnaC-GkDnaI helicase-loader complex

EntireName: GkDnaC-GkDnaI helicase-loader complex
Components
  • Sample: GkDnaC-GkDnaI helicase-loader complex
  • Protein or peptide: GkDnaC-GkDnaI complex

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Supramolecule #1000: GkDnaC-GkDnaI helicase-loader complex

SupramoleculeName: GkDnaC-GkDnaI helicase-loader complex / type: sample / ID: 1000
Oligomeric state: One homohexamer of GkDnaC binds to one dimer (or two monomers) of GkDnaI
Number unique components: 2
Molecular weightTheoretical: 378 KDa

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Macromolecule #1: GkDnaC-GkDnaI complex

MacromoleculeName: GkDnaC-GkDnaI complex / type: protein_or_peptide / ID: 1
Oligomeric state: one GkDnaC hexamer + two GkDnaI monomer (or one GkDnaI dimer)
Recombinant expression: Yes
Source (natural)Organism: Geobacillus kaustophilus (bacteria) / Strain: HTA426
Molecular weightTheoretical: 378 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21DE3

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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

Concentration0.02 mg/mL
BufferpH: 8.5
Details: 20 mM Tris-HCl pH 8.5, 200 mM NaCl, 5mM MgCl2, 1 mM beta-mercaptoethanol, 1 mM ATP-gamma-S
StainingType: NEGATIVE
Details: Grids with adsorbed protein floated sequentially on three drops of 0.75 % w/v uranyl formate each for 20 seconds and then left for staining for 1 minute.
GridDetails: 300 mesh Cu grid with thin carbon support, glow discharged in atmosphere
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 298 K
DateMay 26, 2013
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 25 e/Å2
Tilt angle min0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 85658 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 1.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 62000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle max: 50
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsThe particles were semi-automatically selected using EMAN2 package and manually checked.
CTF correctionDetails: CTFFIND3
Final reconstructionResolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: OTHER / Software - Name: Spider, Relion / Number images used: 10214

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Atomic model buiding 1

Initial modelPDB ID:

4nmn


Chain - Chain ID: A
SoftwareName: Chimera
DetailsHomologous models of GkDnaC and GkDnaI using 4NMN Chain A and 4M4W Chain O as templates respectively were docked into the reconstructed EM map.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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