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- PDB-6ixw: pCBH ParM non-polymerisable quadruple mutant -

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Basic information

Entry
Database: PDB / ID: 6ixw
TitlepCBH ParM non-polymerisable quadruple mutant
ComponentspCBH ParM
KeywordsSTRUCTURAL PROTEIN / Actin ParM
Function / homologyActin-like protein, N-terminal / ParM-like / Actin like proteins N terminal domain / Uncharacterized protein
Function and homology information
Biological speciesClostridium botulinum Prevot_594 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.253 Å
AuthorsKoh, F. / Popp, D. / Narita, A. / Robinson, R.C.
CitationJournal: Nat Commun / Year: 2019
Title: The structure of a 15-stranded actin-like filament from Clostridium botulinum.
Authors: Fujiet Koh / Akihiro Narita / Lin Jie Lee / Kotaro Tanaka / Yong Zi Tan / Venkata P Dandey / David Popp / Robert C Robinson /
Abstract: Microfilaments (actin) and microtubules represent the extremes in eukaryotic cytoskeleton cross-sectional dimensions, raising the question of whether filament architectures are limited by protein ...Microfilaments (actin) and microtubules represent the extremes in eukaryotic cytoskeleton cross-sectional dimensions, raising the question of whether filament architectures are limited by protein fold. Here, we report the cryoelectron microscopy structure of a complex filament formed from 15 protofilaments of an actin-like protein. This actin-like ParM is encoded on the large pCBH Clostridium botulinum plasmid. In cross-section, the ~26 nm diameter filament comprises a central helical protofilament surrounded by intermediate and outer layers of six and eight twisted protofilaments, respectively. Alternating polarity of the layers allows for similar lateral contacts between each layer. This filament design is stiffer than the actin filament, and has likely been selected for during evolution to move large cargos. The comparable sizes of microtubule and pCBH ParM filaments indicate that larger filament architectures are not limited by the protomer fold. Instead, function appears to have been the evolutionary driving force to produce broad, complex filaments.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Dec 12, 2018 / Release: Jun 19, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jun 19, 2019Structure modelrepositoryInitial release
1.1Jul 10, 2019Structure modelData collection / Database referencescitation / citation_author_citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: pCBH ParM
B: pCBH ParM
A: pCBH ParM
hetero molecules


Theoretical massNumber of molelcules
Total (without water)120,80111
Polymers119,3983
Non-polymers1,4038
Water0
1
C: pCBH ParM
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,2754
Polymers39,7991
Non-polymers4763
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1010 Å2
ΔGint-24 kcal/mol
Surface area16170 Å2
MethodPISA
2
B: pCBH ParM
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,2513
Polymers39,7991
Non-polymers4522
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area930 Å2
ΔGint-19 kcal/mol
Surface area15910 Å2
MethodPISA
3
A: pCBH ParM
hetero molecules


Theoretical massNumber of molelcules
Total (without water)40,2754
Polymers39,7991
Non-polymers4763
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1020 Å2
ΔGint-27 kcal/mol
Surface area15870 Å2
MethodPISA
Unit cell
γ
α
β
Length a, b, c (Å)164.153, 93.922, 114.699
Angle α, β, γ (deg.)90.000, 131.550, 90.000
Int Tables number5
Space group name H-MC121

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Components

#1: Protein/peptide pCBH ParM


Mass: 39799.254 Da / Num. of mol.: 3 / Mutation: F42D, I46D, S298D, R299D
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium botulinum Prevot_594 (bacteria)
Gene: T258_3831 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0B4W229
#2: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Adenosine diphosphate / Comment: ADP (energy-carrying molecule) *YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg / Magnesium

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.78 Å3/Da / Density % sol: 55.78 %
Crystal growTemperature: 288 K / Method: vapor diffusion, hanging drop / Details: 28% PEG 400 0.1 M Hepes 0.15 M MgCl2 pH 7.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: NSRRC / Beamline: TPS 05A / Wavelength: 1 Å
DetectorType: RAYONIX MX300-HS / Detector: CCD / Date: Oct 12, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 3.25→20 Å / Num. obs: 20481 / % possible obs: 99.9 % / Redundancy: 3.5 % / Biso Wilson estimate: 61.56 Å2 / Rmerge(I) obs: 0.143 / Rpim(I) all: 0.091 / Rrim(I) all: 0.17 / Χ2: 0.943 / Net I/σ(I): 6.8
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2% possible all
3.25-3.313.20.5749950.5620.3880.6960.95199.1
3.31-3.373.20.49710370.6660.3310.5990.92699.5
3.37-3.433.30.4479850.6910.2960.5380.92699.9
3.43-3.53.40.40710450.7320.2640.4870.94899.8
3.5-3.583.40.4059850.7940.2590.4821.038100
3.58-3.663.50.37810230.7830.2390.4481.07199.9
3.66-3.753.50.30910430.8360.1950.3671.037100
3.75-3.853.50.2439850.9060.1520.2871.038100
3.85-3.963.50.22810430.9140.1420.2691.006100
3.96-4.093.50.18510530.9570.1140.2170.993100
4.09-4.233.60.169980.9630.0990.1890.98100
4.23-4.43.60.13410190.9670.0830.1580.998100
4.4-4.63.50.11810110.9780.0740.1391.00799.9
4.6-4.843.60.110210.9820.0620.1170.955100
4.84-5.143.60.09710440.9870.060.1140.911100
5.14-5.533.60.10910270.9820.0670.1280.887100
5.53-6.073.60.11110260.9780.0690.1310.85100
6.07-6.913.60.09110370.9820.0570.1080.782100
6.91-8.593.50.05610480.9950.0350.0660.807100
8.59-203.40.0410560.9950.0260.0480.765100

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Processing

Software
NameVersionClassification
HKL-2000data reduction
HKL-2000data scaling
PHENIX(1.13_2998: ???)refinement
PDB_EXTRACT3.24data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 3.253→19.974 Å / SU ML: 0.25 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 22.55
RfactorNum. reflection% reflection
Rfree0.214 1012 5.11 %
Rwork0.1577 --
Obs0.1607 19789 96.31 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 192.89 Å2 / Biso mean: 60.3091 Å2 / Biso min: 3.42 Å2
Refinement stepCycle: final / Resolution: 3.253→19.974 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms8156 0 86 0 8242
Biso mean--61.52 --
Num. residues----1024
LS refinement shell

Refinement-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 7

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.253-3.42370.27021200.2062269238983
3.4237-3.6370.28391040.19112652275693
3.637-3.91590.25091460.16882720286698
3.9159-4.30640.20891480.143727772925100
4.3064-4.92140.18351750.123627432918100
4.9214-6.16990.18131520.159127982950100
6.1699-19.97450.21361670.156728182985100

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