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- EMDB-0310: Structure of McrBC without DNA binding domains (one half of the f... -

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Basic information

Entry
Database: EMDB / ID: EMD-0310
TitleStructure of McrBC without DNA binding domains (one half of the full complex)
Map data
SampleMcrB and McrC complex without DNA binding domains
  • 5-methylcytosine-specific restriction enzyme B
  • Protein McrC
  • (ligand) x 3
Function / homology
Function and homology information


restriction endodeoxyribonuclease activity / Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters / DNA catabolic process / DNA restriction-modification system / ATPase activity / GTPase activity / GTP binding / DNA binding / ATP binding
ATPase, dynein-related, AAA domain / P-loop containing nucleoside triphosphate hydrolase / Protein of unknown function DUF3578 / 5-methylcytosine restriction system component / 5-methylcytosine restriction system component, bacterial / AAA+ ATPase domain
5-methylcytosine-specific restriction enzyme B / Protein McrC
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
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.
Validation ReportPDB-ID: 6hz4

SummaryFull reportAbout validation report
History
DepositionOct 22, 2018-
Header (metadata) releaseJul 24, 2019-
Map releaseJul 24, 2019-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6hz4
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0310.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 240 pix.
= 252. Å
1.05 Å/pix.
x 240 pix.
= 252. Å
1.05 Å/pix.
x 240 pix.
= 252. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.24584462 - 0.35694763
Average (Standard dev.)0.0007722372 (±0.010761768)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 251.99998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z252.000252.000252.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.2460.3570.001

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Supplemental data

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Segmentation: #1

Fileemd_0310_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_0310_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #2

Fileemd_0310_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire McrB and McrC complex without DNA binding domains

EntireName: McrB and McrC complex without DNA binding domains
Details: The N-terminal DNA binding domain of McrB is truncated
Number of components: 6

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Component #1: protein, McrB and McrC complex without DNA binding domains

ProteinName: McrB and McrC complex without DNA binding domains
Details: The N-terminal DNA binding domain of McrB is truncated
Recombinant expression: No
MassTheoretical: 510 kDa
SourceSpecies: Escherichia coli K-12 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: BL21-AI

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Component #2: protein, 5-methylcytosine-specific restriction enzyme B

ProteinName: 5-methylcytosine-specific restriction enzyme B / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 35.758492 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #3: protein, Protein McrC

ProteinName: Protein McrC / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 40.643625 kDa
SourceSpecies: Escherichia coli (strain K12) (bacteria) / Strain: K12
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: ligand, PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER

LigandName: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.522196 kDa

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #6: ligand, GUANOSINE-5'-DIPHOSPHATE

LigandName: GUANOSINE-5'-DIPHOSPHATE / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.443201 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 4 mg/mL / pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.5 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 3326

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 225201
3D reconstructionSoftware: RELION / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Target criteria: Cross-correlation coefficient / Refinement space: REAL / Overall bvalue: 74
Output model

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