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- EMDB-20866: Cryo-EM structure of the Thermococcus gammatolerans McrBC complex -

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Basic information

Entry
Database: EMDB / ID: EMD-20866
TitleCryo-EM structure of the Thermococcus gammatolerans McrBC complex
Map dataFL-TgMcrBC
Sample
  • Organelle or cellular component: Complex of the full-length Thermococcus gammatolerans McrBC
    • Protein or peptide: GTPase subunit of restriction endonuclease
    • Protein or peptide: McrBC 5-methylcytosine restriction system component
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: water
KeywordsEndonuclease / AAA protein / GTPase / Methylation-dependent restriction / DNA BINDING PROTEIN
Function / homology
Function and homology information


endonuclease activity / ATP hydrolysis activity / ATP binding / metal ion binding
Similarity search - Function
Type IV methyl-directed restriction enzyme EcoKMcrBC / McrBC 5-methylcytosine restriction system component / : / 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
McrBC 5-methylcytosine restriction system component / GTPase subunit of restriction endonuclease
Similarity search - Component
Biological speciesThermococcus gammatolerans (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.44 Å
AuthorsNiu Y / Suzuki H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM120242 United States
CitationJournal: Nat Commun / Year: 2020
Title: Structural asymmetry governs the assembly and GTPase activity of McrBC restriction complexes.
Authors: Yiming Niu / Hiroshi Suzuki / Christopher J Hosford / Thomas Walz / Joshua S Chappie /
Abstract: McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The GTP-specific AAA + protein McrB powers translocation along DNA and its hydrolysis ...McrBC complexes are motor-driven nucleases functioning in bacterial self-defense by cleaving foreign DNA. The GTP-specific AAA + protein McrB powers translocation along DNA and its hydrolysis activity is stimulated by its partner nuclease McrC. Here, we report cryo-EM structures of Thermococcus gammatolerans McrB and McrBC, and E. coli McrBC. The McrB hexamers, containing the necessary catalytic machinery for basal GTP hydrolysis, are intrinsically asymmetric. This asymmetry directs McrC binding so that it engages a single active site, where it then uses an arginine/lysine-mediated hydrogen-bonding network to reposition the asparagine in the McrB signature motif for optimal catalytic function. While the two McrBC complexes use different DNA-binding domains, these contribute to the same general GTP-recognition mechanism employed by all G proteins. Asymmetry also induces distinct inter-subunit interactions around the ring, suggesting a coordinated and directional GTP-hydrolysis cycle. Our data provide insights into the conserved molecular mechanisms governing McrB family AAA + motors.
History
DepositionOct 29, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseOct 21, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ut5
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20866.png

Downloads & links

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Map

FileDownload / File: emd_20866.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFL-TgMcrBC
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1 Å/pix.
x 400 pix.
= 400. Å		1 Å/pix.
x 400 pix.
= 400. Å		1 Å/pix.
x 400 pix.
= 400. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.07010121 - 0.21496187
Average (Standard dev.)-0.0000029631938 (±0.0033013527)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 400.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z400.000400.000400.000
α/β/γ90.00090.00090.000
start NX/NY/NZ9482110
NX/NY/NZ11313776
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0700.215-0.000

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Supplemental data

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Mask #1

Fileemd_20866_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_20866_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_20866_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of the full-length Thermococcus gammatolerans McrBC

EntireName: Complex of the full-length Thermococcus gammatolerans McrBC
Components
  • Organelle or cellular component: Complex of the full-length Thermococcus gammatolerans McrBC
    • Protein or peptide: GTPase subunit of restriction endonuclease
    • Protein or peptide: McrBC 5-methylcytosine restriction system component
  • Ligand: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
  • Ligand: water

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Supramolecule #1: Complex of the full-length Thermococcus gammatolerans McrBC

SupramoleculeName: Complex of the full-length Thermococcus gammatolerans McrBC
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Thermococcus gammatolerans (archaea)

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Macromolecule #1: GTPase subunit of restriction endonuclease

MacromoleculeName: GTPase subunit of restriction endonuclease / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus gammatolerans (archaea)
Molecular weightTheoretical: 71.668867 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MENQLFIIGI GTGTDEYENF EETILKGVKR NELEGQIGPD ILDNCCSDVC YFWGRSKETI YEKKIDKGDM VLFYVGKRIS RNKVDLNQE TAVYLGIICE TVEISENDVS FLNDFWRKGE NFRFLMFFKK KPEKLHHSIN EINSKLGYNP DYFPIAGYVK P ERMSGVYD ...String:
MENQLFIIGI GTGTDEYENF EETILKGVKR NELEGQIGPD ILDNCCSDVC YFWGRSKETI YEKKIDKGDM VLFYVGKRIS RNKVDLNQE TAVYLGIICE TVEISENDVS FLNDFWRKGE NFRFLMFFKK KPEKLHHSIN EINSKLGYNP DYFPIAGYVK P ERMSGVYD ILKNILKKRG ILKESDSMNE SAGHNIKEDY FRVDMLLNKK GQVILYGPPG TGKTWIARKY VVEETNEKTP GN KWEFITF HQSYSYEEFI EGFRPRTDNE EKIRYVVEDG IFKKIALRAL VKGLFELEDA TIGKDKIHRL YILLTKKEPL SPT EYEEYL RLKRYLWELV GGLPKDKLKN LTPKFYLIID EINRGNISKI FGELITLLEK DKRLGGENQL IVRLPYSGEP FAVP PNLYI IGTMNTADRS IALLDVALRR RFAFIEVEPR PEFLEKENLK KIREKKLKTE DRKRLNEKLN ELFSKLGNDN YFLKT LLEK INVRITVVKD RDHRIGHSYF LNVETVEDLH HVWYYEVLPL LMEYFYNDWE TIKWVLNEKG KEHGNVFFEK LRLTGP NGE EAYQLKVLEG DAFIGALKRI ISKNTPSQEG GATTNEENSP ENTQSQTEGD

UniProtKB: GTPase subunit of restriction endonuclease

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Macromolecule #2: McrBC 5-methylcytosine restriction system component

MacromoleculeName: McrBC 5-methylcytosine restriction system component / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermococcus gammatolerans (archaea)
Molecular weightTheoretical: 54.339406 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPRLTTITLY EHDEKRYRDI AGDKKAIQDA LIKLNKQFKK DFKKLDRSED NSDTEDTIDE SKGVVEVYAN KIKARHYVGF AAVDNVFLQ ILPKVFKPKK EQTQETQEDT WEPILAFIRM LDMAYGLKIK DHDLAYLQGR NLRPNLYEVF IYLFAKSLWS E VQRGYHRE ...String:
MPRLTTITLY EHDEKRYRDI AGDKKAIQDA LIKLNKQFKK DFKKLDRSED NSDTEDTIDE SKGVVEVYAN KIKARHYVGF AAVDNVFLQ ILPKVFKPKK EQTQETQEDT WEPILAFIRM LDMAYGLKIK DHDLAYLQGR NLRPNLYEVF IYLFAKSLWS E VQRGYHRE YVEVHREEKF LRGKLLMSRQ IRKLPHQLNT FSVEVHELIE DNLLNRIFYA SVREALRRTT WGLNRKLLGE LM LAFDGIT PIHLRTEHFE RVHFTRLNER FRRPFELAKL LFMPASGKGR SREVSGFFVD MNKLFERFIE RVLVRNLPPE YKL FYQESY PFLKNQNGSS QKPDYVVRKG NTPVVVLDAK YRELKERIPS SDMLRQLYVY SRIWGYKTSH ENDSKPPAVI VIPS SSTYN QGLPDKPLEF EFFDERKLFI VAYNMDYVKT GAIFKADKNF RRSLNNIIGK LNT

UniProtKB: McrBC 5-methylcytosine restriction system component

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Macromolecule #3: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

MacromoleculeName: 5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE / type: ligand / ID: 3 / Number of copies: 5 / Formula: GSP
Molecular weightTheoretical: 539.246 Da
Chemical component information

ChemComp-GSP:
5'-GUANOSINE-DIPHOSPHATE-MONOTHIOPHOSPHATE

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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: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

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

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 28 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration16.0 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 2078 / Average exposure time: 10.0 sec. / Average electron dose: 8.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
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

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.44 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.6) / Number images used: 226846
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. 2.4.0)
FSC plot (resolution estimation)

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