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Entry
Database: PDB / ID: 4blf
TitleVariable internal flexibility characterizes the helical capsid formed by Agrobacterium VirE2 protein on single-stranded DNA.
ComponentsSINGLE-STRAND DNA-BINDING PROTEIN
KeywordsDNA BINDING PROTEIN / TCOMPLEX / 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 TUMEFACIENS (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 20 Å
AuthorsBharat, T.A.M. / Zbaida, D. / Eisenstein, M. / Frankenstein, Z. / Mehlman, T. / Weiner, L. / Sorzano, C.O.S. / Barak, Y. / Albeck, S. / Briggs, J.A.G. ...Bharat, T.A.M. / Zbaida, D. / Eisenstein, M. / Frankenstein, Z. / Mehlman, T. / Weiner, L. / Sorzano, C.O.S. / Barak, Y. / Albeck, S. / Briggs, J.A.G. / Wolf, S.G. / 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
DepositionMay 2, 2013Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 26, 2013Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2013Group: Database references
Revision 1.2Aug 30, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_image_scans / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id ..._em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

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Assembly

Deposited unit
A: SINGLE-STRAND DNA-BINDING PROTEIN


Theoretical massNumber of molelcules
Total (without water)26,6681
Polymers26,6681
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein SINGLE-STRAND DNA-BINDING PROTEIN / / 63.5 KDA VIRULENCE PROTEIN / VIRE2 PROTEIN


Mass: 26667.943 Da / Num. of mol.: 1 / Fragment: N-TERMINAL DOMAIN, RESIDUES 112-337
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) AGROBACTERIUM TUMEFACIENS (bacteria) / Production host: ESCHERICHIA COLI (E. coli) / References: UniProt: P08062

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: CRYOEM RECONSTRUCTION OF THE AGROBACTERIUM T-COMPLEX / Type: COMPLEX
Details: MICROGRAPHS IN WHICH THON RINGS WERE VISIBLE BEYOND 13 ANGSTROEMS WERE SELECTED.
Buffer solutionName: 50 MM TRIS, 500 MM NACL / pH: 8 / Details: 50 MM TRIS, 500 MM NACL
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 95, INSTRUMENT- HOMEMADE PLUNGER

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Jun 6, 2008
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Nominal defocus max: 3200 nm / Nominal defocus min: 1000 nm / Cs: 2 mm
Image recordingElectron dose: 20 e/Å2 / Film or detector model: GENERIC TVIPS
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1fitPDB2EMmodel fitting
2Bsoft3D reconstruction
3EMAN3D reconstruction
4IHRSR3D reconstruction
5SPIDER3D reconstruction
6Xmipp3D reconstruction
CTF correctionDetails: PHASE-FLIPPING
3D reconstructionResolution: 20 Å / Num. of particles: 8019 / Nominal pixel size: 4.32 Å / Actual pixel size: 4.32 Å
Details: PARTICLES WERE PRE-SELECTED USING XMIPP, AND RECONSTRUCTION WAS CARRIED OUT USING IHRSR PROGRAM IMPLEMENTED IN THE SPIDER PACKAGE. THIS ATOMIC STRUCTURE WAS FITTED INTO THE CRYOEM ENVELOPE ...Details: PARTICLES WERE PRE-SELECTED USING XMIPP, AND RECONSTRUCTION WAS CARRIED OUT USING IHRSR PROGRAM IMPLEMENTED IN THE SPIDER PACKAGE. THIS ATOMIC STRUCTURE WAS FITTED INTO THE CRYOEM ENVELOPE USING FITPDB2EM. SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2339. (DEPOSITION ID: 11520).
Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Cross-correlation coefficient / Details: METHOD--RIGID BODY REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 3BTP

3btp

RefinementHighest resolution: 20 Å
Refinement stepCycle: LAST / Highest resolution: 20 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1863 0 0 0 1863

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