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- EMDB-32114: Structure of McrBC (stalkless mutant) -

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Basic information

Entry
Database: EMDB / ID: EMD-32114
TitleStructure of McrBC (stalkless mutant)
Map dataThis is the B-factor sharpened map
Sample
  • Complex: McrBC (stalkless mutant)
    • Protein or peptide: 5-methylcytosine-specific restriction enzyme B
    • Protein or peptide: Protein McrC
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsAAA+ protein / GTPase / endonuclease / McrBC / stalkless mutant / DNA BINDING PROTEIN
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: 4.5 Å
AuthorsSaikrishnan K / Adhav VA
Funding support1 items
OrganizationGrant numberCountry
Not funded
Citation
Journal: To Be Published
Title: Structure of McrBC (stalkless mutant)
Authors: Saikrishnan K / Adhav VA / 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 KR / Amunts A / Saikrishnan K
History
DepositionOct 27, 2021-
Header (metadata) releaseFeb 15, 2023-
Map releaseFeb 15, 2023-
UpdateJun 26, 2024-
Current statusJun 26, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32114.png

Downloads & links

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Map

FileDownload / File: emd_32114.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is the B-factor sharpened map
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.018
Minimum - Maximum-0.024994975 - 0.06128802
Average (Standard dev.)-0.0000578434 (±0.0039618844)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: This is the unfiltered half-map 2

Fileemd_32114_half_map_1.map
AnnotationThis is the unfiltered half-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: This is the unfiltered half-map 1

Fileemd_32114_half_map_2.map
AnnotationThis is the unfiltered half-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : McrBC (stalkless mutant)

EntireName: McrBC (stalkless mutant)
Components
  • Complex: McrBC (stalkless mutant)
    • Protein or peptide: 5-methylcytosine-specific restriction enzyme B
    • Protein or peptide: Protein McrC
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: McrBC (stalkless mutant)

SupramoleculeName: McrBC (stalkless mutant) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 720 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)
Molecular weightTheoretical: 54.272113 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MESIQPWIEK FIKQAQQQRS QSTKDYPTSY RNLRVKLSFG YGNFTSIPWF AFLGEGQEAS NGIYPVILYY KDFDELVLAY GISDTNEPH AQWQFSSDIP KTIAEYFQAT SGVYPKKYGQ SYYACSQKVS QGIDYTRFAS MLDNIINDYK LIFNSGKSVI P PMSKTESY ...String:
MESIQPWIEK FIKQAQQQRS QSTKDYPTSY RNLRVKLSFG YGNFTSIPWF AFLGEGQEAS NGIYPVILYY KDFDELVLAY GISDTNEPH AQWQFSSDIP KTIAEYFQAT SGVYPKKYGQ SYYACSQKVS QGIDYTRFAS MLDNIINDYK LIFNSGKSVI P PMSKTESY CLEDALNDLF IPETTIETIL KRLTIKKNII LQGPPGVGKT FVARRLAYLL TGEKAPQRVN MVQFHQSYSY ED FIQGYRP NGVGFRRKDG IFYNFCQQAK EQPEKKYIFI IDEINRANLS KVFGEVMMLM EHDKRGENWS VPLTYSENDE ERF YVPENV YIIGLMNTAD RSLAVVDYAL RRRFSFIDIE PGFDTPQFRN FLLNKKAEPS FVESLCQKMN ELNQEISKEA TILG KGFRI GHSYFCCGLE DGTSPDTQWL NEIVMTDIAP LLEEYFFDDP YKQQKWTNKL LGDSSGSHHH HHH

UniProtKB: Type IV methyl-directed restriction enzyme EcoKMcrB subunit

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Macromolecule #2: Protein McrC

MacromoleculeName: Protein McrC / type: protein_or_peptide / ID: 2
Details: Residues from 60 to 99 in database P15006 are replaced by GG
Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)
Molecular weightTheoretical: 36.22352 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MEQPVIPVRN IYYMLTYAWG YLQEIKQANL EAIPGNNLLD ILGYVLNKGV LQLSRRGLEG GNEDTLANRI IKSTLAILIK HEKLNSTIR DEARSLYRKL PGISTLHLTP QHFSYLNGGK NTRYYKFVIS VCKFIVNNSI PGQNKGHYRF YDFERNEKEM S LLYQKFLY ...String:
MEQPVIPVRN IYYMLTYAWG YLQEIKQANL EAIPGNNLLD ILGYVLNKGV LQLSRRGLEG GNEDTLANRI IKSTLAILIK HEKLNSTIR DEARSLYRKL PGISTLHLTP QHFSYLNGGK NTRYYKFVIS VCKFIVNNSI PGQNKGHYRF YDFERNEKEM S LLYQKFLY EFCRRELTSA NTTRSYLKWD ASSISDQSLN LLPRMETDIT IRSSEKILIV DAKYYKSIFS RRMGTEKFHS QN LYQLMNY LWSLKPENGE NIGGLLIYPH VDTAVKHRYK INGFDIGLCT VNLGQEWPCI HQELLDIFDE YLK

UniProtKB: Type IV methyl-directed restriction enzyme EcoKMcrBC, Type IV methyl-directed restriction enzyme EcoKMcrBC

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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

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMTris-ClTris-Cl
50.0 mMKCLPotassium chloride
5.0 mMMgCl2Magnesium chloride
1.0 mMDTTDithiothreitol
GridModel: Quantifoil R3.5/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR
Details: The grid was floated with carbon before glow discharge
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 289 K / Instrument: FEI VITROBOT MARK IV
Details: The sample was blot for 3.0 sec before plunging into liquid ethane.
DetailsThe 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

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 2071 / Average exposure time: 60.0 sec. / Average electron dose: 27.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 131363
Startup modelType of model: OTHER
Details: Initial model was generated using SGD built in Relion
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 60954
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6hz4


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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