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- PDB-7vsr: Structure of McrBC (stalkless mutant) -

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Basic information

Entry
Database: PDB / ID: 7vsr
TitleStructure of McrBC (stalkless mutant)
Components
  • 5-methylcytosine-specific restriction enzyme B
  • Protein McrC
KeywordsDNA BINDING PROTEIN / AAA+ protein / GTPase / endonuclease / McrBC / stalkless mutant
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
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: 4.5 Å
AuthorsSaikrishnan, K. / Adhav, V.A. / Bose, S. / Kutti R, V.
Funding support1items
OrganizationGrant numberCountry
Not funded
Citation
Journal: To Be Published
Title: Structure of McrBC (stalkless mutant)
Authors: Saikrishnan, K. / Adhav, V.A. / Bose, S. / Kutti R, V.
#1: Journal: Nat Commun / Year: 2019
Title: Structure-based mechanism for activation of the AAA+ GTPase McrB by the endonuclease McrC.
Authors: Nirwan, N. / Itoh, Y. / Singh, P. / Bandyopadhyay, S. / Vinothkumar, K.R. / Amunts, A. / Saikrishnan, K.
History
DepositionOct 27, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 15, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
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
M: Protein McrC
N: Protein McrC
hetero molecules


Theoretical massNumber of molelcules
Total (without water)726,99126
Polymers723,71214
Non-polymers3,27912
Water00
1


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

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Components

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


Mass: 54272.113 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: mcrB, rglB, b4346, JW5871 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): AI
References: UniProt: P15005, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters
#2: Protein Protein McrC


Mass: 36223.520 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Residues from 60 to 99 in database P15006 are replaced by GG
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Gene: mcrC, b4345, JW5789 / Production host: Escherichia coli BL21 (bacteria) / Variant (production host): AI, Ai / References: UniProt: P15006
#3: Chemical
ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER


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 / Feature type: SUBJECT OF INVESTIGATION
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: McrBC (stalkless mutant) / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.72 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMTris-ClTris-Cl1
250 mMPotassium chlorideKCL1
35 mMMagnesium chlorideMgCl21
41 mMDithiothreitolDTT1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The McrBC complex was incubated with 2.5mM of GMP-PNP in the presence of 10mM Tris-Cl pH 8.0, 50mM KCl, 5mM MgCl2, 1mM DTT
Specimen supportDetails: The grid was floated with carbon before glow discharge
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R3.5/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K
Details: The sample was blot for 3.0 sec before plunging into liquid ethane

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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 / Nominal magnification: 75000 X / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 27.7 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2071
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION3.1particle selectionAutopick
2EPU2.11image acquisition
4CTFFIND4.1CTF correction
7UCSF Chimera1.13.1model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
19PHENIX1.19.2_4158-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 131363
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60954 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 6HZ4

6hz4


Accession code: 6HZ4 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00434186
ELECTRON MICROSCOPYf_angle_d0.67146208
ELECTRON MICROSCOPYf_dihedral_angle_d13.48413002
ELECTRON MICROSCOPYf_chiral_restr0.0414898
ELECTRON MICROSCOPYf_plane_restr0.0055986

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