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- PDB-6ut3: X-ray structure of Thermococcus gammatolerans McrB AAA+ domain he... -

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Basic information

Entry
Database: PDB / ID: 6ut3
TitleX-ray structure of Thermococcus gammatolerans McrB AAA+ domain hexamer in P21 symmetry
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)
MethodX-RAY DIFFRACTION / SYNCHROTRON / SAD / Resolution: 2.95 Å
AuthorsNiu, Y. / Hosford, C.J. / Chappie, J.S.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI) 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 1.1Dec 2, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Mar 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

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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)303,07814
Polymers300,8236
Non-polymers2,2548
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area26080 Å2
ΔGint-167 kcal/mol
Surface area93880 Å2
Unit cell
Length a, b, c (Å)102.474, 109.619, 120.782
Angle α, β, γ (deg.)90.000, 108.335, 90.000
Int Tables number4
Space group name H-MP1211
Space group name HallP2yb
Symmetry operation#1: x,y,z
#2: -x,y+1/2,-z

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Components

#1: Protein
GTPase subunit of restriction endonuclease


Mass: 50137.219 Da / Num. of mol.: 6 / Fragment: UNP Residues 186-613
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermococcus gammatolerans (strain DSM 15229 / JCM 11827 / EJ3) (archaea)
Strain: DSM 15229 / JCM 11827 / EJ3 / Gene: TGAM_0453 / Plasmid: PET15BP / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): DE3 / References: UniProt: C5A3Z3
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-GSP / 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE


Mass: 539.246 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3S / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: 2.14 Å3/Da / Density % sol: 42.54 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 6.5
Details: 0.1M sodium acetate, pH 6.5, 17.5 % MPD with a drop size of 2 micro-liters and reservoir volume of 650 micro-liters.

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 24-ID-E / Wavelength: 0.9791 Å
DetectorType: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Jun 14, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9791 Å / Relative weight: 1
ReflectionResolution: 2.95→114.65 Å / Num. obs: 57659 / % possible obs: 99.6 % / Redundancy: 6.8 % / Biso Wilson estimate: 111.98 Å2 / CC1/2: 0.999 / Rmerge(I) obs: 0.073 / Rrim(I) all: 0.079 / Net I/σ(I): 14.2
Reflection shellResolution: 2.95→3.04 Å / Redundancy: 6.9 % / Rmerge(I) obs: 1.415 / Mean I/σ(I) obs: 1 / Num. unique obs: 4037 / CC1/2: 0.556 / Rrim(I) all: 1.674 / % possible all: 99.8

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Processing

Software
NameVersionClassification
PHENIX1.14_3260refinement
XDSBUILT=20180307data scaling
CRANK2phasing
BUCCANEERmodel building
XDSBUILT=20180307data reduction
RefinementMethod to determine structure: SAD / Resolution: 2.95→114.65 Å / SU ML: 0.722 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 46.2557
RfactorNum. reflection% reflectionSelection details
Rfree0.3629 1833 3.44 %1439
Rwork0.3452 ---
obs0.3458 53315 99.52 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 127.63 Å2
Refinement stepCycle: LAST / Resolution: 2.95→114.65 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms15827 0 132 0 15959
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00816275
X-RAY DIFFRACTIONf_angle_d1.302222106
X-RAY DIFFRACTIONf_chiral_restr0.09032520
X-RAY DIFFRACTIONf_plane_restr0.00652814
X-RAY DIFFRACTIONf_dihedral_angle_d16.05855857
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.95-3.030.47761390.45223898X-RAY DIFFRACTION99.07
3.03-3.120.46711410.4533970X-RAY DIFFRACTION99.49
3.12-3.220.46281390.48093913X-RAY DIFFRACTION99.83
3.22-3.330.45681410.44173958X-RAY DIFFRACTION99.73
3.33-3.470.4411400.43113922X-RAY DIFFRACTION99.19
3.47-3.630.4121410.41253965X-RAY DIFFRACTION99.76
3.63-3.820.40211410.38963955X-RAY DIFFRACTION99.93
3.82-4.060.38451420.37453965X-RAY DIFFRACTION99.52
4.06-4.370.34831400.34673938X-RAY DIFFRACTION99.56
4.37-4.810.29431410.31033982X-RAY DIFFRACTION99.45
4.81-5.510.35051420.31533990X-RAY DIFFRACTION99.9
5.51-6.940.36851410.34773960X-RAY DIFFRACTION98.99
6.94-114.650.33781450.30224066X-RAY DIFFRACTION99.43

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