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- EMDB-31964: Cryo-EM structure of Arabidopsis DCL3 in complex with a 30-bp RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-31964
TitleCryo-EM structure of Arabidopsis DCL3 in complex with a 30-bp RNA
Map data
Sample
  • Complex: DCL3 in complex with a 30-bp RNA
    • Complex: DCL3
      • Protein or peptide: Dicer-like 3DCL1
    • Complex: RNA
      • RNA: TAS1a RNA forward strand (5'-phosphorylated)
      • RNA: TAS1a RNA reverse strand
  • Ligand: CALCIUM IONCalcium
  • Ligand: ZINC ION
Function / homology
Function and homology information


regulatory ncRNA-mediated post-transcriptional gene silencing / ribonuclease III activity / RNA processing / defense response to virus / nucleic acid binding / ATP binding / nucleus
Similarity search - Function
Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain / PAZ domain profile. ...Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain / PAZ domain profile. / Ribonuclease III, endonuclease domain superfamily / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsWang Q / Du J
CitationJournal: Science / Year: 2021
Title: Mechanism of siRNA production by a plant Dicer-RNA complex in dicing-competent conformation.
Authors: Qian Wang / Yan Xue / Laixing Zhang / Zhenhui Zhong / Suhua Feng / Changshi Wang / Lifan Xiao / Zhenlin Yang / C Jake Harris / Zhe Wu / Jixian Zhai / Maojun Yang / Sisi Li / Steven E Jacobsen / Jiamu Du /
Abstract: In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are a central part of sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24- ...In eukaryotes, small RNAs (sRNAs) play critical roles in multiple biological processes. Dicer endonucleases are a central part of sRNA biogenesis. In plants, DICER-LIKE PROTEIN 3 (DCL3) produces 24-nucleotide (nt) small interfering RNAs (siRNAs) that determine the specificity of the RNA-directed DNA methylation pathway. Here, we determined the structure of a DCL3–pre-siRNA complex in an active dicing-competent state. The 5′-phosphorylated A1 of the guide strand and the 1-nt 3′ overhang of the complementary strand are specifically recognized by a positively charged pocket and an aromatic cap, respectively. The 24-nt siRNA length dependence relies on the separation between the 5′-phosphorylated end of the guide RNA and dual cleavage sites formed by the paired ribonuclease III domains. These structural studies, complemented by functional data, provide insight into the dicing principle for Dicers in general.
History
DepositionSep 14, 2021-
Header (metadata) releaseOct 27, 2021-
Map releaseOct 27, 2021-
UpdateDec 15, 2021-
Current statusDec 15, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0305
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0305
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7vg3
  • Surface level: 0.0305
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31964.png

Downloads & links

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Map

FileDownload / File: emd_31964.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.0305 / Movie #1: 0.0305
Minimum - Maximum-0.07586577 - 0.13531053
Average (Standard dev.)0.00047705055 (±0.005138671)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 168.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.840.840.84
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z168.000168.000168.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0760.1350.000

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Supplemental data

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Sample components

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Entire : DCL3 in complex with a 30-bp RNA

EntireName: DCL3 in complex with a 30-bp RNA
Components
  • Complex: DCL3 in complex with a 30-bp RNA
    • Complex: DCL3
      • Protein or peptide: Dicer-like 3DCL1
    • Complex: RNA
      • RNA: TAS1a RNA forward strand (5'-phosphorylated)
      • RNA: TAS1a RNA reverse strand
  • Ligand: CALCIUM IONCalcium
  • Ligand: ZINC ION

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Supramolecule #1: DCL3 in complex with a 30-bp RNA

SupramoleculeName: DCL3 in complex with a 30-bp RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightExperimental: 200 KDa

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Supramolecule #2: DCL3

SupramoleculeName: DCL3 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3

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Macromolecule #1: Dicer-like 3

MacromoleculeName: Dicer-like 3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 182.176766 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MWSHPQFEKG GGARGGSGGG SWSHPQFEKG FDYKDDDDKG SGSENLYFQG SMHSSLEPEK MEEGGGSNSL KRKFSEIDGD QNLDSVSSP MMTDSNGSYE LKVYEVAKNR NIIAVLGTGI DKSEITKRLI KAMGSSDTDK RLIIFLAPTV NLVKQQCCEI R ALVNLKVE ...String:
MWSHPQFEKG GGARGGSGGG SWSHPQFEKG FDYKDDDDKG SGSENLYFQG SMHSSLEPEK MEEGGGSNSL KRKFSEIDGD QNLDSVSSP MMTDSNGSYE LKVYEVAKNR NIIAVLGTGI DKSEITKRLI KAMGSSDTDK RLIIFLAPTV NLVKQQCCEI R ALVNLKVE EYFGAKGVDK WTSQRWDEEF SKHDVLVMTP QILLDVLRSA FLKLEMVCLL IIDECHHTTG NHPYAKLMKE FY HESTSKP KIFGLTASAV IRKAQVSELE RLMDSKIFNP EEREGVEKFA TTVKEGPILY NPSPSCSLEL KEKLETSHLK FDA SLRRLQ ELGKDSFLNM DNKFETYQKR LSIDYREILH CLDNLGLICA HLAAEVCLEK ISDTKEESET YKECSMVCKE FLED ILSTI GVYLPQDDKS LVDLQQNHLS AVISGHVSPK LKELFHLLDS FRGDKQKQCL ILVERIITAK VIERFVKKEA SLAYL NVLY LTENNPSTNV SAQKMQIEIP DLFQHGKVNL LFITDVVEEG FQVPDCSCMV CFDLPKTMCS YSQSQKHAKQ SNSKSI MFL ERGNPKQRDH LHDLMRREVL IQDPEAPNLK SCPPPVKNGH GVKEIGSMVI PDSNITVSEE AASTQTMSDP PSRNEQL PP CKKLRLDNNL LQSNGKEKVA SSKSKSSSSA AGSKKRKELH GTTCANALSG TWGENIDGAT FQAYKFDFCC NISGEVYS S FSLLLESTLA EDVGKVEMDL YLVRKLVKAS VSPCGQIRLS QEELVKAKYF QQFFFNGMFG KLFVGSKSQG TKREFLLQT DTSSLWHPAF MFLLLPVETN DLASSATIDW SAINSCASIV EFLKKNSLLD LRDSDGNQCN TSSGQEVLLD DKMEETNLIH FANASSDKN SLEELVVIAI HTGRIYSIVE AVSDSSAMSP FEVDASSGYA TYAEYFNKKY GIVLAHPNQP LMKLKQSHHA H NLLVDFNE EMVVKTEPKA GNVRKRKPNI HAHLPPELLA RIDVPRAVLK SIYLLPSVMH RLESLMLASQ LREEIDCSID NF SISSTSI LEAVTTLTCP ESFSMERLEL LGDSVLKYVA SCHLFLKYPD KDEGQLSRQR QSIISNSNLH RLTTSRKLQG YIR NGAFEP RRWTAPGQFS LFPVPCKCGI DTREVPLDPK FFTENMTIKI GKSCDMGHRW VVSKSVSDCA EALIGAYYVS GGLS ASLHM MKWLGIDVDF DPNLVVEAIN RVSLRCYIPK EDELIELERK IQHEFSAKFL LKEAITHSSL RESYSYERLE FLGDS VLDF LITRHLFNTY EQTGPGEMTD LRSACVNNEN FAQVAVKNNL HTHLQRCATV LETQINDYLM SFQKPDETGR SIPSIQ GPK ALGDVVESIA GALLIDTRLD LDQVWRVFEP LLSPLVTPDK LQLPPYRELN ELCDSLGYFF RVKCSNDGVK AQATIQL QL DDVLLTGDGS EQTNKLALGK AASHLLTQLE KRNISRKTSL GDNQSSMDVN LACNHSDRET LTSETTEIQS IVIPFIGP I NMKKGGPRGT LHEFCKKHLW PMPTFDTSEE KSRTPFEFID GGEKRTSFSS FTSTITLRIP NREAVMYAGE ARPDKKSSF DSAVVELLYE LERRKIVIIQ K

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Macromolecule #2: TAS1a RNA forward strand (5'-phosphorylated)

MacromoleculeName: TAS1a RNA forward strand (5'-phosphorylated) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 9.558729 KDa
SequenceString:
UACAAGCGAA UGAGUCAUUC AUCCUAAGUC

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Macromolecule #3: TAS1a RNA reverse strand

MacromoleculeName: TAS1a RNA reverse strand / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 10.267107 KDa
SequenceString:
GACUUAGGAU GAAUGACUCA UUCGCUUGUA GA

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 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

Concentration1.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTris-HClTristris hydrochloride
100.0 mMNaClSodium chloridesodium chloride
2.0 mMCaCl2calcium chloride
1.0 mMDTTdithiothreitol
VitrificationCryogen name: ETHANE

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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
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 155929

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7vg3:
Cryo-EM structure of Arabidopsis DCL3 in complex with a 30-bp RNA

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