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- PDB-3j4t: Helical model of TubZ-Bt two-stranded filament -

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Basic information

Entry
Database: PDB / ID: 3j4t
TitleHelical model of TubZ-Bt two-stranded filament
ComponentsFtsZ/tubulin-related protein
KeywordsSTRUCTURAL PROTEIN / TubZ / FtsZ-like / tubulin-like / plasmid segregation
Function / homologyplasmid partitioning / Tubulin/FtsZ, GTPase domain superfamily / 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 / Tubulin-like protein TubZ
Function and homology information
Biological speciesBacillus thuringiensis (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 10.8 Å
AuthorsAgard, D.A. / Montabana, E.A.
Citation
Journal: 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.
#1: Journal: Proc Natl Acad Sci U S A / Year: 2010
Title: Filament structure of bacterial tubulin homologue TubZ.
Authors: Christopher H S Aylett / Qing Wang / Katharine A Michie / Linda A Amos / Jan Löwe /
Abstract: Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a ...Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a polymerizing cytomotive filament. Together these components drive newly replicated plasmids to opposite ends of the dividing cell. The Bacillus thuringiensis plasmid pBToxis relies on a filament of the tubulin/FtsZ-like protein TubZ for its segregation. By combining crystallography and electron microscopy, we have determined the structure of this filament. We explain how GTP hydrolysis weakens the subunit-subunit contact and also shed light on the partitioning of the plasmid-adaptor complex. The double helical superstructure of TubZ filaments is unusual for tubulin-like proteins. Filaments of ParM, the actin-like partitioning protein, are also double helical. We suggest that convergent evolution shapes these different types of cytomotive filaments toward a general mechanism for plasmid separation.
History
DepositionOct 4, 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 21, 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_ref_seq_dif
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_database_related.content_type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type / _struct_ref_seq_dif.details

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Structure visualization

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  • Biological unit as representative helical assembly
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Assembly

Deposited unit
F: FtsZ/tubulin-related protein


Theoretical massNumber of molelcules
Total (without water)55,0521
Polymers55,0521
Non-polymers00
Water0
1
F: FtsZ/tubulin-related protein
x 10


Theoretical massNumber of molelcules
Total (without water)550,52410
Polymers550,52410
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation9
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: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 10 / Rise per n subunits: 22.048 Å / Rotation per n subunits: -168.158 °)

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Components

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


Mass: 55052.438 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus thuringiensis (bacteria) / Strain: serovar israelensis / Gene: pBt156 / Plasmid: pET151topoD / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3)star / References: UniProt: Q8KNP3
Sequence detailsMUTATION L2V IS INHERITED FROM PDB ENTRY 2XKA. THE SAMPLE IMAGED FOR THIS ENTRY CONTAINS NO MUTATIONS.

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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: TubZ was preheated at 37 degrees and polymerization was initiated with saturating GTPgammaS. TubZ was then incubated for 30 seconds at room temperature before sample application, 4.5 second ...Details: TubZ was preheated at 37 degrees and polymerization was initiated with saturating GTPgammaS. TubZ was then incubated for 30 seconds at room temperature before sample application, 4.5 second blotting, and plunge-freezing 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: May 26, 2010
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: 4500 nm / Nominal defocus min: 1500 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: 348

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2SPIDER3D reconstruction
CTF correctionDetails: Whole Micrograph
Helical symmertyAngular rotation/subunit: -168.15849 ° / Axial rise/subunit: 22.04811 Å / Axial symmetry: C1
3D reconstructionMethod: Iterative Helical Real Space Refinement / Resolution: 10.8 Å / Resolution method: FSC 0.143 CUT-OFF / Actual pixel size: 1.203 Å
Details: Final map has been low-pass filtered to 11 Angstrom and high-pass filtered to 35 Angstrom. A B-factor of -452 Angstrom was applied using the program bfactor. A cylindrical mask of radius ~78 ...Details: Final map has been low-pass filtered to 11 Angstrom and high-pass filtered to 35 Angstrom. A B-factor of -452 Angstrom was applied using the program bfactor. A cylindrical mask of radius ~78 Angstrom has been applied.
Symmetry type: HELICAL
Atomic model buildingProtocol: OTHER / Space: REAL
Details: METHOD--Chimera DETAILS--Just local fitting done with Chimera
Atomic model buildingPDB-ID: 2XKA

2xka


Pdb chain-ID: F / Accession code: 2XKA / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms3274 0 0 0 3274

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