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- EMDB-2640: Negative-stain electron microscopy of the human GINS complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2640
TitleNegative-stain electron microscopy of the human GINS complex
Map dataReconstruction of the human GINS complex
Sample
  • Sample: Human GINS complex
  • Protein or peptide: GINS
KeywordsDNA replication / CMG / GINS complex / Psf1 B domain
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 30.0 Å
AuthorsCarroni M / De March M / Krastanova I / Medagli B / Taylor IK / Brick P / Amenitsch H / Patwardhan A / Onesti S
CitationJournal: Sci Rep / Year: 2017
Title: New insights into the GINS complex explain the controversy between existing structural models.
Authors: Marta Carroni / Matteo De March / Barbara Medagli / Ivet Krastanova / Ian A Taylor / Heinz Amenitsch / Hiroyuchi Araki / Francesca M Pisani / Ardan Patwardhan / Silvia Onesti /
Abstract: GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron ...GINS is a key component of eukaryotic replicative forks and is composed of four subunits (Sld5, Psf1, Psf2, Psf3). To explain the discrepancy between structural data from crystallography and electron microscopy (EM), we show that GINS is a compact tetramer in solution as observed in crystal structures, but also forms a double-tetrameric population, detectable by EM. This may represent an intermediate step towards the assembly of two replicative helicase complexes at origins, moving in opposite directions within the replication bubble. Reconstruction of the double-tetrameric form, combined with small-angle X-ray scattering data, allows the localisation of the B domain of the Psf1 subunit in the free GINS complex, which was not visible in previous studies and is essential for the formation of a functional replication fork.
History
DepositionMay 6, 2014-
Header (metadata) releaseJun 11, 2014-
Map releaseJun 17, 2015-
UpdateJul 8, 2015-
Current statusJul 8, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2640.png

Downloads & links

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Map

FileDownload / File: emd_2640.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the human GINS complex
Voxel sizeX=Y=Z: 4 Å
Density
Contour LevelBy AUTHOR: 0.13 / Movie #1: 0.13
Minimum - Maximum-0.1992006 - 0.97747266
Average (Standard dev.)0.00244183 (±0.05716141)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-39-40-40
Dimensions808080
Spacing808080
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z444
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-40-39-40
NC/NR/NS808080
D min/max/mean-0.1990.9770.002

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Supplemental data

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Sample components

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Entire : Human GINS complex

EntireName: Human GINS complex
Components
  • Sample: Human GINS complex
  • Protein or peptide: GINS

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Supramolecule #1000: Human GINS complex

SupramoleculeName: Human GINS complex / type: sample / ID: 1000
Details: The sample was in equilibrium with a monomeric form (single GINS tetramer)
Oligomeric state: dimer of human GINS tetramers / Number unique components: 1
Molecular weightExperimental: 200 KDa / Theoretical: 200 KDa
Method: SEC-MALLS (size-exclusion chromatography coupled with multi angle laser light scattering)

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Macromolecule #1: GINS

MacromoleculeName: GINS / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Oligomeric state: dimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Tissue: all / Cell: all / Organelle: nucleus / Location in cell: nucleus
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Codon Plus / Recombinant plasmid: pProEX-Hta

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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

Concentration0.05 mg/mL
BufferpH: 7.8 / Details: 20 mM TrisHCl pH 7.8, 150 mM NaCl, 0.5 mM TCEP
StainingType: NEGATIVE
Details: Protein adsorbed on carbon coated grids, stained with potassium phospho tungstate pH 7.0 for 4 minutes.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification
DateDec 20, 2009
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: entire frame
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 30.0 Å / Resolution method: OTHER / Software - Name: IMAGIC, Spider / Number images used: 14390

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Atomic model buiding 1

Initial modelPDB ID:

2eho


Chain - #0 - Chain ID: E / Chain - #1 - Chain ID: F / Chain - #2 - Chain ID: G / Chain - #3 - Chain ID: H / Chain - #4 - Chain ID: I / Chain - #5 - Chain ID: J / Chain - #6 - Chain ID: K / Chain - #7 - Chain ID: L
SoftwareName: Chimera
DetailsA dimer of tetramers observed in the crystal was fitted.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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