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- PDB-3j4s: Helical Model of TubZ-Bt four-stranded filament -

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Basic information

Entry
Database: PDB / ID: 3j4s
TitleHelical Model of TubZ-Bt four-stranded filament
ComponentsFtsZ/tubulin-related protein
KeywordsSTRUCTURAL PROTEIN / FtsZ-like / Tubulin-like / GTPase
Function / homology
Function and homology information


plasmid partitioning / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / GTPase activity / GTP binding / identical protein binding / metal ion binding / cytoplasm
Similarity search - Function
Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / Tubulin-like protein TubZ
Similarity search - Component
Biological speciesBacillus thuringiensis (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsMontabana, E.A. / Agard, D.A.
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Bacterial tubulin TubZ-Bt transitions between a two-stranded intermediate and a four-stranded filament upon GTP hydrolysis.
Authors: Elizabeth A Montabana / David A Agard /
Abstract: Cytoskeletal filaments form diverse superstructures that are highly adapted for specific functions. The recently discovered TubZ subfamily of tubulins is involved in type III plasmid partitioning ...Cytoskeletal filaments form diverse superstructures that are highly adapted for specific functions. The recently discovered TubZ subfamily of tubulins is involved in type III plasmid partitioning systems, facilitating faithful segregation of low copy-number plasmids during bacterial cell division. One such protein, TubZ-Bt, is found on the large pBtoxis plasmid in Bacillus thuringiensis, and interacts via its extended C terminus with a DNA adaptor protein TubR. Here, we use cryo-electron microscopy to determine the structure of TubZ-Bt filaments and light scattering to explore their mechanism of polymerization. Surprisingly, we find that the helical filament architecture is remarkably sensitive to nucleotide state, changing from two-stranded to four-stranded depending on the ability of TubZ-Bt to hydrolyze GTP. We present pseudoatomic models of both the two- and four-protofilament forms based on cryo-electron microscopy reconstructions (10.8 Å and 6.9 Å, respectively) of filaments formed under different nucleotide states. These data lead to a model in which the two-stranded filament is a necessary intermediate along the pathway to formation of the four-stranded filament. Such nucleotide-directed structural polymorphism is to our knowledge an unprecedented mechanism for the formation of polar filaments.
History
DepositionOct 3, 2013Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2014Provider: repository / Type: Initial release
Revision 1.1Mar 5, 2014Group: Database references
Revision 1.2Mar 19, 2014Group: Database references
Revision 1.3Jul 18, 2018Group: Author supporting evidence / Data collection / Category: em_single_particle_entity / em_software / Item: _em_software.image_processing_id
Revision 1.4Feb 28, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_database_related / pdbx_initial_refinement_model / pdbx_struct_oper_list / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

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  • Biological unit as representative helical assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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  • Superimposition on EM map
  • EMDB-5762
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: FtsZ/tubulin-related protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)54,8792
Polymers54,4361
Non-polymers4431
Water0
1
A: FtsZ/tubulin-related protein
hetero molecules
x 24


Theoretical massNumber of molelcules
Total (without water)1,317,09648
Polymers1,306,45924
Non-polymers10,63724
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation23
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 4 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 24 / Rise per n subunits: 43.545 Å / Rotation per n subunits: 31.788 °)

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Components

#1: Protein FtsZ/tubulin-related protein / TubZ-Bt


Mass: 54435.785 Da / Num. of mol.: 1 / Mutation: L2V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus thuringiensis (bacteria) / Strain: serovar israelensis / Gene: pBt156 / Plasmid: modified pET151topoD / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)star / References: UniProt: Q8KNP3
#2: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: TubZ-Bt / Type: COMPLEX
Buffer solutionName: HMK100 / pH: 7.7
Details: 100 mM potassium acetate, 5 mM magnesium acetate, 50 mM HEPES
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh copper grid with holey carbon support, glow discharged
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 %
Details: Add GTP to grow filaments for 6 minutes before application to grid. Blot 4.5 seconds before plunging into liquid ethane (FEI VITROBOT MARK III).
Method: Blot 4.5 s before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Apr 22, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 62000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 800 nm / Cs: 2.2 mm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: Oxford side-entry cryo stage
Image recordingElectron dose: 20 e/Å2 / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Details: 8K x 8K
Image scansNum. digital images: 414
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1MDFFmodel fitting
2SPIDER3D reconstruction
CTF correctionDetails: Whole Micrograph
Helical symmertyAngular rotation/subunit: 31.78843 ° / Axial rise/subunit: 43.54512 Å / Axial symmetry: C4
3D reconstructionMethod: Iterative Helical Real Space Refinement / Resolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Actual pixel size: 0.94 Å
Details: Final map has been low-pass filtered to 7 Angstrom and high-pass filtered to 30 Angstrom. A B-factor of -309 Angstrom was applied using the program bfactor. A cylindrical mask of radius ~80 ...Details: Final map has been low-pass filtered to 7 Angstrom and high-pass filtered to 30 Angstrom. A B-factor of -309 Angstrom was applied using the program bfactor. A cylindrical mask of radius ~80 Angstrom has been applied.
Symmetry type: HELICAL
Atomic model buildingSpace: REAL
Details: DETAILS--Initial local fitting was done using Chimera and then Molecular Dynamics Flexible Fitting (MDFF) was used for flexible fitting.
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
12XKA

2xka

F2XKA1
22XKB

2xkb

C2XKB2
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms3277 0 28 0 3305

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