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Basic information

Entry
Database: EMDB / ID: EMD-2339
TitleVariable internal flexibility characterizes the helical capsid formed by Agrobacterium VirE2 protein on single-stranded DNA.
Map dataCryoEM reconstruction of the Agrobacterium T-complex
Sample
  • Sample: Agrobacterium T-complex
  • Protein or peptide: VirE2
  • DNA: short oligomeric 26mer DNA
Keywordstcomplex / agrobacterium / helical reconstruction
Function / homologyVirE2 / VirE2 / DNA-mediated transformation / host cell nucleus / DNA binding / extracellular region / identical protein binding / Single-strand DNA-binding protein
Function and homology information
Biological speciesAgrobacterium fabrum str. C58 (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsBharat TAM / Zbaida D / Eisenstein M / Frankenstein Z / Mehlman T / Weiner L / Sorzano COS / Barak Y / Albeck S / Briggs JAG ...Bharat TAM / Zbaida D / Eisenstein M / Frankenstein Z / Mehlman T / Weiner L / Sorzano COS / Barak Y / Albeck S / Briggs JAG / Wolf SG / Elbaum M
CitationJournal: Structure / Year: 2013
Title: Variable internal flexibility characterizes the helical capsid formed by agrobacterium VirE2 protein on single-stranded DNA.
Authors: Tanmay A M Bharat / David Zbaida / Miriam Eisenstein / Ziv Frankenstein / Tevie Mehlman / Lev Weiner / Carlos Oscar S Sorzano / Yoav Barak / Shira Albeck / John A G Briggs / Sharon G Wolf / Michael Elbaum /
Abstract: Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 ...Agrobacterium is known for gene transfer to plants. In addition to a linear ssDNA oligonucleotide, Agrobacterium tumefaciens secretes an abundant ssDNA-binding effector, VirE2. In many ways VirE2 adapts the conjugation mechanism to transform the eukaryotic host. The crystal structure of VirE2 shows two compact domains joined by a flexible linker. Bound to ssDNA, VirE2 forms an ordered solenoidal shell, or capsid known as the T-complex. Here, we present a three-dimensional reconstruction of the VirE2-ssDNA complex using cryo-electron microscopy and iterative helical real-space reconstruction. High-resolution refinement was not possible due to inherent heterogeneity in the protein structure. By a combination of computational modeling, chemical modifications, mass spectroscopy, and electron paramagnetic resonance, we found that the N-terminal domain is tightly constrained by both tangential and longitudinal links, while the C terminus is weakly constrained. The quaternary structure is thus rigidly assembled while remaining locally flexible. This flexibility may be important in accommodating substrates without sequence specificity.
History
DepositionMar 25, 2013-
Header (metadata) releaseApr 24, 2013-
Map releaseJun 26, 2013-
UpdateJul 17, 2013-
Current statusJul 17, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4blf
  • Surface level: 0.008
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4blf
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2339.jpg

Downloads & links

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Map

FileDownload / File: emd_2339.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM reconstruction of the Agrobacterium T-complex
Voxel sizeX=Y=Z: 4.32 Å
Density
Contour LevelBy AUTHOR: 0.008 / Movie #1: 0.008
Minimum - Maximum-0.0447155 - 0.05394139
Average (Standard dev.)0.00051121 (±0.01020005)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.324.324.32
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
α/β/γ90.00090.00090.000
start NX/NY/NZ-27-15-36
NX/NY/NZ553173
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-0.0450.0540.001

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Supplemental data

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Sample components

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Entire : Agrobacterium T-complex

EntireName: Agrobacterium T-complex
Components
  • Sample: Agrobacterium T-complex
  • Protein or peptide: VirE2
  • DNA: short oligomeric 26mer DNA

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Supramolecule #1000: Agrobacterium T-complex

SupramoleculeName: Agrobacterium T-complex / type: sample / ID: 1000 / Oligomeric state: Helical / Number unique components: 2

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Macromolecule #1: VirE2

MacromoleculeName: VirE2 / type: protein_or_peptide / ID: 1 / Oligomeric state: Helical / Recombinant expression: Yes
Source (natural)Organism: Agrobacterium fabrum str. C58 (bacteria)
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: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Agrobacterium fabrum str. C58 (bacteria)

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration1 mg/mL
BufferpH: 8 / Details: 50 mM Tris, 500 mM NaCl
GridDetails: Quantifoil holey carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: HOMEMADE PLUNGER
DetailsProtein was mixed with 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: 3.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at high-magnification (>100,000)
DateJun 6, 2008
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS / Average electron dose: 20 e/Å2 / Details: Image data was collected as focal pairs.
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: 14.67 Å
Applied symmetry - Helical parameters - Δ&Phi: 110.09 °
Resolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Bsoft, EMAN, Xmipp, Spider, IHRSR
DetailsParticles were picked and preselected using routines of Xmipp, and then reconstruction was carried out using IHRSR.

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Atomic model buiding 1

Initial modelPDB ID:

3btp


Chain - Chain ID: A
SoftwareName: fitPDB2EM
DetailsThe N and C terminal domain were fit separately by exhaustive molecular modeling using the fitPDB2EM program. Only the N-terminal domain could be constrained strongly.
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: Highest cross-correlation
Output model

PDB-4blf:
Variable internal flexibility characterizes the helical capsid formed by Agrobacterium VirE2 protein on single-stranded DNA.

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