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- EMDB-20869: Body 1 for multi-body refinement of the McrB-AAA+ and McrC comple... -

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Basic information

Entry
Database: EMDB / ID: EMD-20869
TitleBody 1 for multi-body refinement of the McrB-AAA+ and McrC complex from Thermococcus gammatolerans
Map dataBody1
Sample
  • Organelle or cellular component: Tetradecameric assembly of the McrB-AAA_McrC complex from T. gammatolerans
Biological speciesThermococcus gammatolerans (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.95 Å
AuthorsNiu Y / Suzuki H / Hosford CJ / Chappie JS / Walz T
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM120242 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-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20869.png

Downloads & links

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Map

FileDownload / File: emd_20869.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBody1
Voxel sizeX=Y=Z: 1.25 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.10052439 - 0.16192809
Average (Standard dev.)0.0000510311 (±0.005207944)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.251.251.25
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
start NX/NY/NZ79740
NX/NY/NZ93103213
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1010.1620.000

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Supplemental data

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Mask #1

Fileemd_20869_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_20869_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_20869_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 : Tetradecameric assembly of the McrB-AAA_McrC complex from T. gamm...

EntireName: Tetradecameric assembly of the McrB-AAA_McrC complex from T. gammatolerans
Components
  • Organelle or cellular component: Tetradecameric assembly of the McrB-AAA_McrC complex from T. gammatolerans

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Supramolecule #1: Tetradecameric assembly of the McrB-AAA_McrC complex from T. gamm...

SupramoleculeName: Tetradecameric assembly of the McrB-AAA_McrC complex from T. gammatolerans
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Thermococcus gammatolerans (archaea)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration14.0 mg/mL
BufferpH: 7.5
GridModel: C-flat-1.2/1.3 / Material: COPPER / 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
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: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 4271 / Average exposure time: 10.0 sec. / Average electron dose: 8.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.8)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 2.4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 204593
FSC plot (resolution estimation)

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