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- EMDB-2368: Negative-staining electron microscopy structure of the Agrobacter... -

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Entry
Database: EMDB / ID: EMD-2368
TitleNegative-staining electron microscopy structure of the Agrobacterium tumefaciens T-complex, composed of the protein VirE2 coating single-stranded DNA.
Map dataNegative-staining EM reconstruction of the Agrobacterium T-complex
Sample
  • Sample: Negative-staining recosntruction of the Agrobacterium T-complex
  • Protein or peptide: Agrobacterium tumefaciens VirE2 protein
  • DNA: short oligomeric 26mer DNA
KeywordsT-complex / agrobacterium / helical reconstruction / negative staining
Biological speciesAgrobacterium fabrum str. C58 (bacteria)
Methodhelical reconstruction / negative staining / Resolution: 25.0 Å
AuthorsAbu-Arish A / Frenkiel-Krispin D / Fricke T / Tzfira T / Citovsky V / Wolf SG / Elbaum M
CitationJournal: J Biol Chem / Year: 2004
Title: Three-dimensional reconstruction of Agrobacterium VirE2 protein with single-stranded DNA.
Authors: Asmahan Abu-Arish / Daphna Frenkiel-Krispin / Tobin Fricke / Tzvi Tzfira / Vitaly Citovsky / Sharon Grayer Wolf / Michael Elbaum /
Abstract: Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the ...Agrobacterium tumefaciens infects plant cells by a unique mechanism involving an interkingdom genetic transfer. A single-stranded DNA substrate is transported across the two cell walls along with the bacterial virulence proteins VirD2 and VirE2. A single VirD2 molecule covalently binds to the 5'-end of the single-stranded DNA, while the VirE2 protein binds stoichiometrically along the length of the DNA, without sequence specificity. An earlier transmission/scanning transmission electron microscopy study indicated a solenoidal ("telephone coil") organization of the VirE2-DNA complex. Here we report a three-dimensional reconstruction of this complex using electron microscopy and single-particle image-processing methods. We find a hollow helical structure of 15.7-nm outer diameter, with a helical rise of 51.5 nm and 4.25 VirE2 proteins/turn. The inner face of the protein units contains a continuous wall and an inward protruding shelf. These structures appear to accommodate the DNA binding. Such a quaternary arrangement naturally sequesters the DNA from cytoplasmic nucleases and suggests a mechanism for its nuclear import by decoration with host cell factors. Coexisting with the helices, we also found VirE2 tetrameric ring structures. A two-dimensional average of the latter confirms the major features of the three-dimensional reconstruction.
History
DepositionApr 23, 2013-
Header (metadata) releaseMay 29, 2013-
Map releaseJun 5, 2013-
UpdateJun 5, 2013-
Current statusJun 5, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0175
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2368.jpg

Downloads & links

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Map

FileDownload / File: emd_2368.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative-staining EM reconstruction of the Agrobacterium T-complex
Voxel sizeX=Y=Z: 3.2 Å
Density
Contour LevelBy AUTHOR: 0.0175 / Movie #1: 0.0175
Minimum - Maximum-0.00776185 - 0.04504754
Average (Standard dev.)0.00569092 (±0.0096853)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 256.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.23.23.2
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z256.000256.000256.000
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0080.0450.006

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Supplemental data

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Sample components

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Entire : Negative-staining recosntruction of the Agrobacterium T-complex

EntireName: Negative-staining recosntruction of the Agrobacterium T-complex
Components
  • Sample: Negative-staining recosntruction of the Agrobacterium T-complex
  • Protein or peptide: Agrobacterium tumefaciens VirE2 protein
  • DNA: short oligomeric 26mer DNA

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Supramolecule #1000: Negative-staining recosntruction of the Agrobacterium T-complex

SupramoleculeName: Negative-staining recosntruction of the Agrobacterium T-complex
type: sample / ID: 1000 / Details: Helical assembly / Oligomeric state: Helical / Number unique components: 2

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Macromolecule #1: Agrobacterium tumefaciens VirE2 protein

MacromoleculeName: Agrobacterium tumefaciens VirE2 protein / type: protein_or_peptide / ID: 1
Details: Negative-staining EM reconstruction of the Agrobacterium T-complex
Oligomeric state: Helical / Recombinant expression: Yes
Source (natural)Organism: Agrobacterium fabrum str. C58 (bacteria)
Molecular weightTheoretical: 58 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #2: short oligomeric 26mer DNA

MacromoleculeName: short oligomeric 26mer DNA / type: dna / ID: 2 / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Agrobacterium fabrum str. C58 (bacteria)

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Experimental details

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Structure determination

Methodnegative staining
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

StainingType: NEGATIVE / Details: 1.5% Uranyl acetate staining
GridDetails: Carbon coated grids
VitrificationCryogen name: NONE / Instrument: OTHER
DetailsProtein was mixed with M13 circular single-stranded DNA.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.6 µm
Sample stageSpecimen holder model: OTHER
DateJan 1, 2004
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase-flipping
Final reconstructionApplied symmetry - Helical parameters - Δz: 12.11 Å
Applied symmetry - Helical parameters - Δ&Phi: 84.7 °
Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Software - Name: Bsoft, Spider, IHRSR
DetailsHelical reconstruction using IHRSR

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