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- EMDB-7814: Human nuclear exosome-MTR4 RNA complex - focused reconstruction o... -

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Basic information

Entry
Database: EMDB / ID: EMD-7814
TitleHuman nuclear exosome-MTR4 RNA complex - focused reconstruction on the DIS3-RNBCS domains
Map dataFocused reconstruction of DIS3-RNBCS domains within a human nuclear exosome-MTR4 complex
Sample
  • Complex: Human nuclear exosome-MTR4 helicase captured after unwinding a DNA/RNA substrate
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWeick E-M / Lima CD
CitationJournal: Cell / Year: 2018
Title: Helicase-Dependent RNA Decay Illuminated by a Cryo-EM Structure of a Human Nuclear RNA Exosome-MTR4 Complex.
Authors: Eva-Maria Weick / M Rhyan Puno / Kurt Januszyk / John C Zinder / Michael A DiMattia / Christopher D Lima /
Abstract: The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4- ...The ribonucleolytic RNA exosome interacts with RNA helicases to degrade RNA. To understand how the 3' to 5' Mtr4 helicase engages RNA and the nuclear exosome, we reconstituted 14-subunit Mtr4-containing RNA exosomes from Saccharomyces cerevisiae, Schizosaccharomyces pombe, and human and show that they unwind structured substrates to promote degradation. We loaded a human exosome with an optimized DNA-RNA chimera that stalls MTR4 during unwinding and determined its structure to an overall resolution of 3.45 Å by cryoelectron microscopy (cryo-EM). The structure reveals an RNA-engaged helicase atop the non-catalytic core, with RNA captured within the central channel and DIS3 exoribonuclease active site. MPP6 tethers MTR4 to the exosome through contacts to the RecA domains of MTR4. EXOSC10 remains bound to the core, but its catalytic module and cofactor C1D are displaced by RNA-engaged MTR4. Competition for the exosome core may ensure that RNA is committed to degradation by DIS3 when engaged by MTR4.
History
DepositionApr 22, 2018-
Header (metadata) releaseMay 9, 2018-
Map releaseJun 20, 2018-
UpdateJun 27, 2018-
Current statusJun 27, 2018Processing 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
  • Download
  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7814.png

Downloads & links

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Map

FileDownload / File: emd_7814.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused reconstruction of DIS3-RNBCS domains within a human nuclear exosome-MTR4 complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesY (Sec.)X (Row.)Z (Col.)
1.07 Å/pix.
x 384 pix.
= 410.88 Å		1.07 Å/pix.
x 384 pix.
= 410.88 Å		1.07 Å/pix.
x 384 pix.
= 410.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.14451362 - 0.20910871
Average (Standard dev.)0.000034779656 (±0.0016653091)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 410.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z410.880410.880410.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ384384384
MAP C/R/S312
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.1450.2090.000

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Supplemental data

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Sample components

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Entire : Human nuclear exosome-MTR4 helicase captured after unwinding a DN...

EntireName: Human nuclear exosome-MTR4 helicase captured after unwinding a DNA/RNA substrate
Components
  • Complex: Human nuclear exosome-MTR4 helicase captured after unwinding a DNA/RNA substrate

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Supramolecule #1: Human nuclear exosome-MTR4 helicase captured after unwinding a DN...

SupramoleculeName: Human nuclear exosome-MTR4 helicase captured after unwinding a DNA/RNA substrate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#15
Details: Human C1D/Rrp47 also in the sample, but was not observed in density
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 690 KDa

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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

Concentration1.0 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris-HCl
100.0 mMsodium chlorideNaCl
0.5 mMmagnesium chlorideMgCl2
2.0 mMAMPPNP
1.0 mMBME
0.05 %CHAPSO
GridModel: Quantifoil R2/2 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.037 kPa
VitrificationCryogen name: ETHANE / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: 30 sec wait time, 2.5 sec blot time.
DetailsSample was monodisperse upon elution from gel filtration prior to vitrification.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 1439 / Average electron dose: 85.23 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 278185
CTF correctionSoftware:
Namedetails
Gctf (ver. 1.06)determine
RELION (ver. 2.1.b1)apply
Startup modelType of model: NONE / Details: Ab initio model determination
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1b1) / Details: Focused refinement on DIS3-RNBCS / Number images used: 91483
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 0.6.1) / Software - details: from 0.6.1 to 0.6.5
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1b1)
Final 3D classificationNumber classes: 8 / Avg.num./class: 122284 / Software - Name: RELION (ver. 2.1b1)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModels were rebuilt manually using Coot, with real space refinement with local scaling followed by Phenix real space refinement with suboptimal global scaling.
RefinementSpace: REAL / Protocol: BACKBONE TRACE / Overall B value: 90.6 / Target criteria: Correlation coefficient

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