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- EMDB-0311: Structure of McrBC without DNA binding domains (Class 1) -

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Basic information

Entry
Database: EMDB / ID: EMD-0311
TitleStructure of McrBC without DNA binding domains (Class 1)
Map data
Sample
  • Complex: McrB and McrC complex without DNA binding domains
    • Protein or peptide: 5-methylcytosine-specific restriction enzyme B
    • Protein or peptide: Protein McrC
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
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
Type IV methyl-directed restriction enzyme EcoKMcrB subunit / Type IV methyl-directed restriction enzyme EcoKMcrBC
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli (strain K12) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.2 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.
History
DepositionOct 22, 2018-
Header (metadata) releaseJul 24, 2019-
Map releaseJul 24, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6hz5
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0311.png

Downloads & links

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Map

FileDownload / File: emd_0311.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum-0.046531003 - 0.103662156
Average (Standard dev.)0.00065788446 (±0.0047758766)
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-0.0470.1040.001

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Supplemental data

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Mask #1

Fileemd_0311_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_0311_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_0311_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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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
Components
  • Complex: McrB and McrC complex without DNA binding domains
    • Protein or peptide: 5-methylcytosine-specific restriction enzyme B
    • Protein or peptide: Protein McrC
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: McrB and McrC complex without DNA binding domains

SupramoleculeName: McrB and McrC complex without DNA binding domains / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: The N-terminal DNA binding domain of McrB is truncated
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21(AI)
Molecular weightTheoretical: 510 KDa

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Macromolecule #1: 5-methylcytosine-specific restriction enzyme B

MacromoleculeName: 5-methylcytosine-specific restriction enzyme B / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 35.758492 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKTESYCLE DALNDLFIPE TTIETILKRL TIKKNIILQG PPGVGKTFVA RRLAYLLTGE KAPQRVNMVQ FHQSYSYEDF IQGYRPNGV GFRRKDGIFY NFCQQAKEQP EKKYIFIIDE INRANLSKVF GEVMMLMEHD KRGENWSVPL TYSENDEERF Y VPENVYII ...String:
MSKTESYCLE DALNDLFIPE TTIETILKRL TIKKNIILQG PPGVGKTFVA RRLAYLLTGE KAPQRVNMVQ FHQSYSYEDF IQGYRPNGV GFRRKDGIFY NFCQQAKEQP EKKYIFIIDE INRANLSKVF GEVMMLMEHD KRGENWSVPL TYSENDEERF Y VPENVYII GLMNTADRSL AVVDYALRRR FSFIDIEPGF DTPQFRNFLL NKKAEPSFVE SLCQKMNELN QEISKEATIL GK GFRIGHS YFCCGLEDGT SPDTQWLNEI VMTDIAPLLE EYFFDDPYKQ QKWTNKLLGD SSGSHHHHHH

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Macromolecule #2: Protein McrC

MacromoleculeName: Protein McrC / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria) / Strain: K12
Molecular weightTheoretical: 40.643625 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEQPVIPVRN IYYMLTYAWG YLQEIKQANL EAIPGNNLLD ILGYVLNKGV LQLSRRGLEL DYNPNTEIIP GIKGRIEFAK TIRGFHLNH GKTVSTFDML NEDTLANRII KSTLAILIKH EKLNSTIRDE ARSLYRKLPG ISTLHLTPQH FSYLNGGKNT R YYKFVISV ...String:
MEQPVIPVRN IYYMLTYAWG YLQEIKQANL EAIPGNNLLD ILGYVLNKGV LQLSRRGLEL DYNPNTEIIP GIKGRIEFAK TIRGFHLNH GKTVSTFDML NEDTLANRII KSTLAILIKH EKLNSTIRDE ARSLYRKLPG ISTLHLTPQH FSYLNGGKNT R YYKFVISV CKFIVNNSIP GQNKGHYRFY DFERNEKEMS LLYQKFLYEF CRRELTSANT TRSYLKWDAS SISDQSLNLL PR METDITI RSSEKILIVD AKYYKSIFSR RMGTEKFHSQ NLYQLMNYLW SLKPENGENI GGLLIYPHVD TAVKHRYKIN GFD IGLCTV NLGQEWPCIH QELLDIFDEY LK

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Macromolecule #3: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / type: ligand / ID: 3 / Number of copies: 6 / Formula: GNP
Molecular weightTheoretical: 522.196 Da
Chemical component information

ChemComp-GNP:
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / GppNHp, GMPPNP, energy-carrying molecule analogue*YM / 5'-Guanylyl imidodiphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 6 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration4.0 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
0.1 MNaClSodium chloridesodium chloride
10.0 mMTris-HClTristris(hydroxymethyl)aminomethane hydrochloride buffer
5.0 mMMgCl2Magnesium chloride
2.0 mMC10H17N6O13P35'-Guanylyl imidodiphosphate
GridModel: C-flat-2/2 4C / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 5.0 µm / Calibrated defocus min: 0.3 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 2-20 / Number grids imaged: 1 / Number real images: 3326 / Average exposure time: 8.0 sec. / Average electron dose: 1.5 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 771763
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.1.0)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1.0)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1.0) / Number images used: 225201
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 74 / Target criteria: Cross-correlation coefficient
Output model

PDB-6hz5:
Structure of McrBC without DNA binding domains (Class 1)

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