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- EMDB-27827: RNA-free Human Dis3L2 -

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ID or keywords:

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Basic information

Entry
Database: EMDB / ID: EMD-27827
TitleRNA-free Human Dis3L2
Map data
Sample
  • Complex: Inactive RNA-free HsDis3L2 (D391N)
    • Protein or peptide: DIS3-like exonuclease 2
Keywords3'-5' exonuclease / RNA-free exonuclease / human exonuclease / RNA BIND PROTEIN-RNA complex / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


polyuridylation-dependent mRNA catabolic process / miRNA catabolic process / mitotic sister chromatid separation / : / Z-decay: degradation of maternal mRNAs by zygotically expressed factors / poly(U) RNA binding / stem cell population maintenance / : / mRNA catabolic process / RNA nuclease activity ...polyuridylation-dependent mRNA catabolic process / miRNA catabolic process / mitotic sister chromatid separation / : / Z-decay: degradation of maternal mRNAs by zygotically expressed factors / poly(U) RNA binding / stem cell population maintenance / : / mRNA catabolic process / RNA nuclease activity / P-body / mitotic cell cycle / Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters / 3'-5'-RNA exonuclease activity / negative regulation of cell population proliferation / cell division / magnesium ion binding / cytoplasm / cytosol
Similarity search - Function
DIS3-like exonuclease 2 / DIS3-like exonuclease 2, C-terminal / DIS3-like exonuclease 2 C terminal / Rrp44-like cold shock domain / Rrp44-like cold shock domain / Dis3-like cold-shock domain 2 / Dis3-like cold-shock domain 2 (CSD2) / Ribonuclease II/R, conserved site / Ribonuclease II family signature. / Ribonuclease II/R ...DIS3-like exonuclease 2 / DIS3-like exonuclease 2, C-terminal / DIS3-like exonuclease 2 C terminal / Rrp44-like cold shock domain / Rrp44-like cold shock domain / Dis3-like cold-shock domain 2 / Dis3-like cold-shock domain 2 (CSD2) / Ribonuclease II/R, conserved site / Ribonuclease II family signature. / Ribonuclease II/R / RNB domain / RNB / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DIS3-like exonuclease 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsMeze K / Thomas DR / Joshua-Tor L
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01-GM114147 United States
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: A shape-shifting nuclease unravels structured RNA.
Authors: Katarina Meze / Armend Axhemi / Dennis R Thomas / Ahmet Doymaz / Leemor Joshua-Tor /
Abstract: RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts. Dis3-like 2 (Dis3L2) is a 3'-5' exoribonuclease that plays a critical role in human development. ...RNA turnover pathways ensure appropriate gene expression levels by eliminating unwanted transcripts. Dis3-like 2 (Dis3L2) is a 3'-5' exoribonuclease that plays a critical role in human development. Dis3L2 independently degrades structured substrates, including coding and noncoding 3' uridylated RNAs. While the basis for Dis3L2's substrate recognition has been well characterized, the mechanism of structured RNA degradation by this family of enzymes is unknown. We characterized the discrete steps of the degradation cycle by determining cryogenic electron microscopy structures representing snapshots along the RNA turnover pathway and measuring kinetic parameters for RNA processing. We discovered a dramatic conformational change that is triggered by double-stranded RNA (dsRNA), repositioning two cold shock domains by 70 Å. This movement exposes a trihelix linker region, which acts as a wedge to separate the two RNA strands. Furthermore, we show that the trihelix linker is critical for dsRNA, but not single-stranded RNA, degradation. These findings reveal the conformational plasticity of Dis3L2 and detail a mechanism of structured RNA degradation.
History
DepositionAug 14, 2022-
Header (metadata) releaseMar 1, 2023-
Map releaseMar 1, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27827.png

Downloads & links

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Map

FileDownload / File: emd_27827.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.66 Å
Density
Contour LevelBy AUTHOR: 0.0763
Minimum - Maximum-0.37936398 - 0.65636814
Average (Standard dev.)-0.00023683859 (±0.01794311)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 198.00002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_27827_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_27827_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Inactive RNA-free HsDis3L2 (D391N)

EntireName: Inactive RNA-free HsDis3L2 (D391N)
Components
  • Complex: Inactive RNA-free HsDis3L2 (D391N)
    • Protein or peptide: DIS3-like exonuclease 2

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Supramolecule #1: Inactive RNA-free HsDis3L2 (D391N)

SupramoleculeName: Inactive RNA-free HsDis3L2 (D391N) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 100 KDa

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Macromolecule #1: DIS3-like exonuclease 2

MacromoleculeName: DIS3-like exonuclease 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Exoribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 96.54332 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSHPDYRMNL RPLGTPRGVS AVAGPHDIGA SPGDKKSKNR STRGKKKSIF ETYMSKEDVS EGLKRGTLIQ GVLRINPKKF HEAFIPSPD GDRDIFIDGV VARNRALNGD LVVVKLLPEE HWKVVKPESN DKETEAAYES DIPEELCGHH LPQQSLKSYN D SPDVIVEA ...String:
MSHPDYRMNL RPLGTPRGVS AVAGPHDIGA SPGDKKSKNR STRGKKKSIF ETYMSKEDVS EGLKRGTLIQ GVLRINPKKF HEAFIPSPD GDRDIFIDGV VARNRALNGD LVVVKLLPEE HWKVVKPESN DKETEAAYES DIPEELCGHH LPQQSLKSYN D SPDVIVEA QFDGSDSEDG HGITQNVLVD GVKKLSVCVS EKGREDGDAP VTKDETTCIS QDTRALSEKS LQRSAKVVYI LE KKHSRAA TGFLKLLADK NSELFRKYAL FSPSDHRVPR IYVPLKDCPQ DFVARPKDYA NTLFICRIVD WKEDCNFALG QLA KSLGQA GEIEPETEGI LTEYGVDFSD FSSEVLECLP QGLPWTIPPE EFSKRRDLRK DCIFTIDPST ARDLNDALSC KPLA DGNFK VGVHIADVSY FVPEGSDLDK VAAERATSVY LVQKVVPMLP RLLCEELCSL NPMSDKLTFS VIWTLTPEGK ILDEW FGRT IIRSCTKLSY EHAQSMIESP TEKIPAKELP PISPEHSSEE VHQAVLNLHG IAKQLRQQRF VDGALRLDQL KLAFTL DHE TGLPQGCHIY EYRESNKLVE EFMLLANMAV AHKIHRAFPE QALLRRHPPP QTRMLSDLVE FCDQMGLPVD FSSAGAL NK SLTQTFGDDK YSLARKEVLT NMCSRPMQMA LYFCSGLLQD PAQFRHYALN VPLYTHFTSP IRRFADVLVH RLLAAALG Y RERLDMAPDT LQKQADHCND RRMASKRVQE LSTSLFFAVL VKESGPLESE AMVMGILKQA FDVLVLRYGV QKRIYCNAL ALRSHHFQKV GKKPELTLVW EPEDMEQEPA QQVITIFSLV EVVLQAESTA LKYSAILK

UniProtKB: DIS3-like exonuclease 2

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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

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMNaClsodium chloride
5.0 mMDTTDithiothreitol
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.042 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3840 pixel / Digitization - Dimensions - Height: 3712 pixel / Digitization - Frames/image: 1-25 / Number grids imaged: 1 / Number real images: 4095 / Average exposure time: 5.0 sec. / Average electron dose: 51.75 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.8000000000000003 µm / Calibrated defocus min: 0.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.6 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 160000
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: 990287
Startup modelType of model: OTHER / Details: Ab Initio in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0.0) / Number images used: 162793
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.0) / Details: stochastic gradient descent
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0.0)
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC (ver. v3.0.0)

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Atomic model buiding 1

Initial modelPDB ID:

4pmw


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsChain A of PDB 4PMW (MmDis3L2) was fit into the map via rigid body fitting. Residues were then mutated to match the human protein and manual building done to correct for differences. Next rounds of refinement and building were done in PHENIX and Coot, respectively, until the final structure was obtained.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8e27:
RNA-free Human Dis3L2

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