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- EMDB-2339: Variable internal flexibility characterizes the helical capsid fo... -
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Basic information
Entry | Database: EMDB / ID: EMD-2339 | |||||||||
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Title | Variable internal flexibility characterizes the helical capsid formed by Agrobacterium VirE2 protein on single-stranded DNA. | |||||||||
![]() | CryoEM reconstruction of the Agrobacterium T-complex | |||||||||
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![]() | tcomplex / agrobacterium / helical reconstruction | |||||||||
Function / homology | VirE2 / VirE2 / DNA-mediated transformation / host cell nucleus / DNA binding / extracellular region / identical protein binding / Single-strand DNA-binding protein![]() | |||||||||
Biological species | ![]() | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 20.0 Å | |||||||||
![]() | Bharat 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 | |||||||||
![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 247.1 KB | ![]() | |
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Header (meta data) | ![]() ![]() | 11.3 KB 11.3 KB | Display Display | ![]() |
Images | ![]() | 75.5 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 4blfMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | CryoEM reconstruction of the Agrobacterium T-complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 4.32 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
-Entire : Agrobacterium T-complex
Entire | Name: Agrobacterium T-complex |
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Components |
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-Supramolecule #1000: Agrobacterium T-complex
Supramolecule | Name: Agrobacterium T-complex / type: sample / ID: 1000 / Oligomeric state: Helical / Number unique components: 2 |
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-Macromolecule #1: VirE2
Macromolecule | Name: VirE2 / type: protein_or_peptide / ID: 1 / Oligomeric state: Helical / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() |
-Macromolecule #2: short oligomeric 26mer DNA
Macromolecule | Name: short oligomeric 26mer DNA / type: dna / ID: 2 / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: No |
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Source (natural) | Organism: ![]() |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | helical reconstruction |
Aggregation state | helical array |
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Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 8 / Details: 50 mM Tris, 500 mM NaCl |
Grid | Details: Quantifoil holey carbon |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: HOMEMADE PLUNGER |
Details | Protein was mixed with single-stranded DNA |
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Electron microscopy
Microscope | FEI TECNAI F20 |
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Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at high-magnification (>100,000) |
Date | Jun 6, 2008 |
Image recording | Category: CCD / Film or detector model: GENERIC TVIPS / Average electron dose: 20 e/Å2 / Details: Image data was collected as focal pairs. |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | ![]() Model: Tecnai F20 / Image courtesy: FEI Company |
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Image processing
Details | Particles were picked and preselected using routines of Xmipp, and then reconstruction was carried out using IHRSR. |
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Final reconstruction | Applied 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 |
CTF correction | Details: Phase-flipping |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A |
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Software | Name: fitPDB2EM |
Details | The 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. |
Refinement | Space: RECIPROCAL / Protocol: FLEXIBLE FIT / Target criteria: Highest cross-correlation |
Output model | ![]() PDB-4blf: |