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- PDB-6ut4: Cryo-EM structure of the asymmetric AAA+ domain hexamer from Ther... -

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Basic information

Entry
Database: PDB / ID: 6ut4
TitleCryo-EM structure of the asymmetric AAA+ domain hexamer from Thermococcus gammatolerans McrB
ComponentsGTPase subunit of restriction endonuclease
KeywordsDNA BINDING PROTEIN / AAA protein / GTPase / Methylation-dependent restriction
Function / homology
Function and homology information


endonuclease activity / ATP hydrolysis activity / ATP binding / metal ion binding
Similarity search - Function
: / 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
5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / GTPase subunit of restriction endonuclease
Similarity search - Component
Biological speciesThermococcus gammatolerans (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 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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  • EMDB-20865
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Structure viewerMolecule:
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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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)304,20518
Polymers300,8236
Non-polymers3,38112
Water21612
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
GTPase subunit of restriction endonuclease


Mass: 50137.219 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus gammatolerans (archaea) / Production host: Escherichia coli (E. coli) / References: UniProt: C5A3Z3
#2: Chemical
ChemComp-GSP / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE


Mass: 539.246 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H16N5O13P3S / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Homo-hexameric assembly of the AAA domain of Thermococcus gammatolerans McrB
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Thermococcus gammatolerans (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 10 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 FIELD
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: 1599
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.15.2_3472refinement
PHENIX1.15.2_3472refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1.8CTF correction
7Cootmodel fitting
9PHENIX1.15.2model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139306 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004319556
ELECTRON MICROSCOPYf_angle_d0.739926474
ELECTRON MICROSCOPYf_chiral_restr0.052891
ELECTRON MICROSCOPYf_plane_restr0.0063311
ELECTRON MICROSCOPYf_dihedral_angle_d16.779411691

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