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- PDB-6hz6: Structure of McrBC without DNA binding domains (Class 2) -

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Basic information

Entry
Database: PDB / ID: 6hz6
TitleStructure of McrBC without DNA binding domains (Class 2)
Components
  • 5-methylcytosine-specific restriction enzyme B
  • Protein McrC
KeywordsDNA BINDING PROTEIN / AAA+ superfamily / restriction enzyme
Function / homology
Function and homology information


type IV site-specific deoxyribonuclease activity / restriction endodeoxyribonuclease activity / endonuclease complex / double-stranded methylated DNA binding / hemi-methylated DNA-binding / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / DNA restriction-modification system / DNA catabolic process / endonuclease activity / GTPase activity ...type IV site-specific deoxyribonuclease activity / restriction endodeoxyribonuclease activity / endonuclease complex / double-stranded methylated DNA binding / hemi-methylated DNA-binding / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / DNA restriction-modification system / DNA catabolic process / endonuclease activity / GTPase activity / GTP binding / ATP hydrolysis activity / DNA binding / ATP binding / identical protein binding
Similarity search - Function
5-methylcytosine restriction system component, bacterial / Type IV methyl-directed restriction enzyme EcoKMcrBC / McrBC 5-methylcytosine restriction system component / Type IV methyl-directed restriction enzyme EcoKMcrB subunit, DNA-binding domain / MrcB-like, N-terminal domain / 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 / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / Type IV methyl-directed restriction enzyme EcoKMcrB subunit / Type IV methyl-directed restriction enzyme EcoKMcrBC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
Model detailsTwo homo hexamers dimerized by one homo dimer
AuthorsItoh, Y. / Nirwan, N. / Saikrishnan, K. / Amunts, A.
CitationJournal: Nat Commun / Year: 2019
Title: Structure-based mechanism for activation of the AAA+ GTPase McrB by the endonuclease McrC.
Authors: Neha Nirwan / Yuzuru Itoh / Pratima Singh / Sutirtha Bandyopadhyay / Kutti R Vinothkumar / Alexey Amunts / Kayarat Saikrishnan /
Abstract: The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation ...The AAA+ GTPase McrB powers DNA cleavage by the endonuclease McrC. The GTPase itself is activated by McrC. The architecture of the GTPase and nuclease complex, and the mechanism of their activation remained unknown. Here, we report a 3.6 Å structure of a GTPase-active and DNA-binding deficient construct of McrBC. Two hexameric rings of McrB are bridged by McrC dimer. McrC interacts asymmetrically with McrB protomers and inserts a stalk into the pore of the ring, reminiscent of the γ subunit complexed to αβ of F-ATPase. Activation of the GTPase involves conformational changes of residues essential for hydrolysis. Three consecutive nucleotide-binding pockets are occupied by the GTP analogue 5'-guanylyl imidodiphosphate and the next three by GDP, which is suggestive of sequential GTP hydrolysis.
History
DepositionOct 22, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 24, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria

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Assembly

Deposited unit
A: 5-methylcytosine-specific restriction enzyme B
B: 5-methylcytosine-specific restriction enzyme B
C: 5-methylcytosine-specific restriction enzyme B
D: 5-methylcytosine-specific restriction enzyme B
E: 5-methylcytosine-specific restriction enzyme B
F: 5-methylcytosine-specific restriction enzyme B
M: Protein McrC
G: 5-methylcytosine-specific restriction enzyme B
H: 5-methylcytosine-specific restriction enzyme B
I: 5-methylcytosine-specific restriction enzyme B
J: 5-methylcytosine-specific restriction enzyme B
K: 5-methylcytosine-specific restriction enzyme B
L: 5-methylcytosine-specific restriction enzyme B
N: Protein McrC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)516,32732
Polymers510,38914
Non-polymers5,93818
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area67600 Å2
ΔGint-292 kcal/mol
Surface area159850 Å2
MethodPISA

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Components

#1: Protein
5-methylcytosine-specific restriction enzyme B / EcoKMcrBC


Mass: 35758.492 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: mcrB, rglB, b4346, JW5871 / Plasmid: pHis / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(AI)
References: UniProt: P15005, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#2: Protein Protein McrC


Mass: 40643.625 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: mcrC, b4345, JW5789 / Plasmid: pHis / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(AI) / References: UniProt: P15006
#3: Chemical
ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / 5'-Guanylyl imidodiphosphate


Mass: 522.196 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H17N6O13P3 / Feature type: SUBJECT OF INVESTIGATION
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg
#5: Chemical
ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE / Guanosine diphosphate


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM

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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: McrB and McrC complex without DNA binding domains / Type: COMPLEX
Details: The N-terminal DNA binding domain of McrB is truncated
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.51 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(AI)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
10.10 Msodium chlorideNaClSodium chloride1
210 mMtris(hydroxymethyl)aminomethane hydrochloride bufferTris-HClTris1
35.0 mMMagnesium chlorideMgCl21
42.0 mM5'-Guanylyl imidodiphosphateC10H17N6O13P31
SpecimenConc.: 4 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-2/2 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / 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 / Nominal magnification: 130000 X / Calibrated defocus min: 300 nm / Calibrated defocus max: 5000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 1.5 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3326
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 20 / Used frames/image: 2-20

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
4Gctf1.06CTF correction
7Coot0.88 ELmodel fitting
9PHENIXdev-3290model refinement
10RELION2.1.0initial Euler assignment
11RELION2.1.0final Euler assignment
12RELION2.1.0classification
13RELION2.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 771763
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 225201 / Symmetry type: POINT
Atomic model buildingB value: 74 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Cross-correlation coefficient

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