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- PDB-5aey: actin-like ParM protein bound to AMPPNP -

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Basic information

Entry
Database: PDB / ID: 5aey
Titleactin-like ParM protein bound to AMPPNP
ComponentsPLASMID SEGREGATION PROTEIN PARM
KeywordsSTRUCTURAL PROTEIN / BACTERIAL CYTOSKELETON / PLASMID SEGREGATION / ACTIN- LIKE PROTEIN
Function / homology
Function and homology information


plasmid partitioning / identical protein binding
Similarity search - Function
Plasmid segregation protein ParM/StbA / : / Plasmid segregation protein ParM, N-terminal / Plasmid segregation protein ParM, C-terminal / ParM-like / ATPase, nucleotide binding domain
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Plasmid segregation protein ParM
Similarity search - Component
Biological speciesESCHERICHIA COLI (E. coli)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsBharat, T.A.M. / Murshudov, G.N. / Sachse, C. / Lowe, J.
CitationJournal: Nature / Year: 2015
Title: Structures of actin-like ParM filaments show architecture of plasmid-segregating spindles.
Authors: Tanmay A M Bharat / Garib N Murshudov / Carsten Sachse / Jan Löwe /
Abstract: Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with ...Active segregation of Escherichia coli low-copy-number plasmid R1 involves formation of a bipolar spindle made of left-handed double-helical actin-like ParM filaments. ParR links the filaments with centromeric parC plasmid DNA, while facilitating the addition of subunits to ParM filaments. Growing ParMRC spindles push sister plasmids to the cell poles. Here, using modern electron cryomicroscopy methods, we investigate the structures and arrangements of ParM filaments in vitro and in cells, revealing at near-atomic resolution how subunits and filaments come together to produce the simplest known mitotic machinery. To understand the mechanism of dynamic instability, we determine structures of ParM filaments in different nucleotide states. The structure of filaments bound to the ATP analogue AMPPNP is determined at 4.3 Å resolution and refined. The ParM filament structure shows strong longitudinal interfaces and weaker lateral interactions. Also using electron cryomicroscopy, we reconstruct ParM doublets forming antiparallel spindles. Finally, with whole-cell electron cryotomography, we show that doublets are abundant in bacterial cells containing low-copy-number plasmids with the ParMRC locus, leading to an asynchronous model of R1 plasmid segregation.
History
DepositionJan 12, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 22, 2015Provider: repository / Type: Initial release
Revision 1.1May 13, 2015Group: Database references
Revision 1.2Jul 15, 2015Group: Database references
Revision 1.3May 8, 2024Group: Author supporting evidence / Data collection ...Author supporting evidence / Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_image_scans / em_single_particle_entity / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _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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Assembly

Deposited unit
A: PLASMID SEGREGATION PROTEIN PARM
B: PLASMID SEGREGATION PROTEIN PARM
C: PLASMID SEGREGATION PROTEIN PARM
D: PLASMID SEGREGATION PROTEIN PARM
E: PLASMID SEGREGATION PROTEIN PARM
hetero molecules


Theoretical massNumber of molelcules
Total (without water)180,69710
Polymers178,1665
Non-polymers2,5315
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-0.966499, 0.256669, 0.000111), (-0.256669, -0.966499, 0.000469), (0.000228, 0.000425, 1)63.37815, 104.53696, -24.66172
3given(1), (1), (1)
4given(0.868757, -0.495239, 0.000357), (0.495239, 0.868757, -0.000275), (-0.000173, 0.000416, 1)28.85133, -12.73555, -49.35579
5given(1), (1), (1)
6given(-0.712995, 0.701169, -7.0E-6), (-0.701169, -0.712995, -0.000386), (-0.000275, -0.00027, 1)32.2009, 109.51602, -73.99551
7given(1), (1), (1)
8given(0.509133, -0.860688, 0.00034), (0.860688, 0.509132, -0.000544), (0.000295, 0.000569, 1)60.2804, -9.45153, -98.76075
9given(1), (1), (1)
10given(-0.966765, 0.255665, -4.4E-5), (-0.255665, -0.966765, 0.000789), (0.000159, 0.000774, 1)63.46462, 104.47076, -24.75331
11given(1), (1), (1)
12given(0.869075, -0.49468, 0.000337), (0.49468, 0.869075, 0.000803), (-0.00069, -0.000532, 1)28.84354, -12.83636, -49.33437
13given(1), (1), (1)
14given(-0.712983, 0.701181, 4.4E-5), (-0.701181, -0.712983, 0.001046), (0.000765, 0.000715, 0.999999)32.23336, 109.34197, -74.1532
15given(1), (1), (1)
16given(-0.966665, 0.256043, -0.000371), (-0.256043, -0.966665, 8.4E-5), (-0.000337, 0.000176, 1)63.45798, 104.5378, -24.6722
17given(1), (1), (1)
18given(0.868559, -0.495586, -0.000148), (0.495586, 0.868559, -0.000225), (0.00024, 0.000122, 1)28.93916, -12.73243, -49.39465
19given(1), (1), (1)
20given(-0.966497, 0.256678, -0.000136), (-0.256678, -0.966497, 0.000356), (-4.0E-5, 0.000379, 1)63.40269, 104.51842, -24.71951

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Components

#1: Protein
PLASMID SEGREGATION PROTEIN PARM / PARA LOCUS 36 KDA PROTEIN / PROTEIN STBA / PARM


Mass: 35633.223 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Details: PARM WAS INCUBATED WITH AMPPNP / Source: (gene. exp.) ESCHERICHIA COLI (E. coli) / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21 / Variant (production host): AI / References: UniProt: P11904
#2: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*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: PARM BOUND TO AMPPNP / Type: COMPLEX / Details: MICROGRAPHS COLLECTED ON AN FEI KRIOS.
Buffer solutionName: 50 MM TRIS-HCL, 100 MM KCL, AND 1 MM MGCL2 / pH: 7 / Details: 50 MM TRIS-HCL, 100 MM KCL, AND 1 MM MGCL2
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE
Details: VITRIFICATION 1 -- CRYOGEN- ETHANE, HUMIDITY- 100, INSTRUMENT- FEI VITROBOT MARK IV,

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Feb 2, 2014
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 28 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)
Radiation wavelengthRelative weight: 1

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Processing

3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: HELICAL
RefinementHighest resolution: 4.3 Å
Refinement stepCycle: LAST / Highest resolution: 4.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12520 0 155 0 12675

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