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- PDB-6ut7: Fitted model for the tetradecameric assembly of Thermococcus gamm... -

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Basic information

Entry
Database: PDB / ID: 6ut7
TitleFitted model for the tetradecameric assembly of Thermococcus gammatolerans McrB AAA+ hexamers with bound McrC
Components
  • GTPase subunit of restriction endonuclease
  • McrBC 5-methylcytosine restriction system component
KeywordsDNA BINDING PROTEIN / Endonuclease / AAA protein / GTPase / Methylation-dependent restriction
Function / homology
Function and homology information


endonuclease activity / ATP hydrolysis activity / ATP binding
Similarity search - Function
Type IV methyl-directed restriction enzyme EcoKMcrBC / McrBC 5-methylcytosine restriction system component / ATPase, dynein-related, AAA domain / AAA domain (dynein-related subfamily) / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
GUANOSINE-5'-DIPHOSPHATE / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / McrBC 5-methylcytosine restriction system component / GTPase subunit of restriction endonuclease
Similarity search - Component
Biological speciesThermococcus gammatolerans (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.26 Å
AuthorsNiu, Y. / Suzuki, H. / Hosford, C.J. / Chappie, J.S. / Walz, T.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM120242 United States
CitationJournal: Nat Commun / Year: 2020
Title: Structural asymmetry governs the assembly and GTPase activity of McrBC restriction complexes.
Authors: Yiming Niu / Hiroshi Suzuki / Christopher J Hosford / Thomas Walz / Joshua S Chappie /
Abstract: McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The GTP-specific AAA + protein McrB powers translocation along DNA and its hydrolysis ...McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The GTP-specific AAA + protein McrB powers translocation along DNA and its hydrolysis activity is stimulated by its partner nuclease McrC. Here, we report cryo-EM structures of Thermococcus gammatolerans McrB and McrBC, and E. coli McrBC. The McrB hexamers, containing the necessary catalytic machinery for basal GTP hydrolysis, are intrinsically asymmetric. This asymmetry directs McrC binding so that it engages a single active site, where it then uses an arginine/lysine-mediated hydrogen-bonding network to reposition the asparagine in the McrB signature motif for optimal catalytic function. While the two McrBC complexes use different DNA-binding domains, these contribute to the same general GTP-recognition mechanism employed by all G proteins. Asymmetry also induces distinct inter-subunit interactions around the ring, suggesting a coordinated and directional GTP-hydrolysis cycle. Our data provide insights into the conserved molecular mechanisms governing McrB family AAA + motors.
History
DepositionOct 29, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 21, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-20868
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: GTPase subunit of restriction endonuclease
B: GTPase subunit of restriction endonuclease
C: GTPase subunit of restriction endonuclease
D: GTPase subunit of restriction endonuclease
E: GTPase subunit of restriction endonuclease
F: GTPase subunit of restriction endonuclease
G: McrBC 5-methylcytosine restriction system component
H: GTPase subunit of restriction endonuclease
I: GTPase subunit of restriction endonuclease
J: GTPase subunit of restriction endonuclease
K: GTPase subunit of restriction endonuclease
L: GTPase subunit of restriction endonuclease
M: GTPase subunit of restriction endonuclease
N: McrBC 5-methylcytosine restriction system component
hetero molecules


Theoretical massNumber of molelcules
Total (without water)716,70438
Polymers710,32514
Non-polymers6,37824
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: scanning transmission electron microscopy, Negative staining showed dumbbell-shaped particles of the complexes.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
GTPase subunit of restriction endonuclease /


Mass: 50137.219 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus gammatolerans (archaea) / Production host: Escherichia coli (E. coli) / References: UniProt: C5A3Z3
#2: Protein McrBC 5-methylcytosine restriction system component


Mass: 54339.406 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus gammatolerans (archaea) / Production host: Escherichia coli (E. coli) / References: UniProt: C5A3Z2
#3: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Comment: GDP, energy-carrying molecule*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-GSP / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE


Mass: 539.246 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C10H16N5O13P3S
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Tetradecameric assembly of the McrB-AAA_McrC complex from T. gammatolerans
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Thermococcus gammatolerans (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 14 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4271
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.16_3549refinement
PHENIX1.16_3549refinement
EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2SerialEMimage acquisition
4CTFFIND4.1.8CTF correction
7Cootmodel fitting
8PHENIX1.15.2model fitting
9UCSF Chimeramodel fitting
11PHENIX1.15.2model refinement
12RELION3initial Euler assignment
13RELION3final Euler assignment
14cryoSPARC2.4.0classification
15RELION33D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.26 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204593 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 16.53 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004546094
ELECTRON MICROSCOPYf_angle_d0.831762370
ELECTRON MICROSCOPYf_chiral_restr0.05536846
ELECTRON MICROSCOPYf_plane_restr0.00567814
ELECTRON MICROSCOPYf_dihedral_angle_d18.919427642

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