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- EMDB-31182: Cryo-EM structure of Arabidopsis DCL1 in complex with pre-miRNA 166f -

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Basic information

Entry
Database: EMDB / ID: EMD-31182
TitleCryo-EM structure of Arabidopsis DCL1 in complex with pre-miRNA 166f
Map data
Sample
  • Complex: Binary complex of DCL1 in complex with pre-miRNA 166f
    • Protein or peptide: Endoribonuclease Dicer homolog 1
    • RNA: pre-miRNA 166f
Function / homology
Function and homology information


ribonuclease III activity / siRNA processing / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / helicase activity / rRNA processing / nucleic acid binding / DNA binding / RNA binding / ATP binding / metal ion binding ...ribonuclease III activity / siRNA processing / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / helicase activity / rRNA processing / nucleic acid binding / DNA binding / RNA binding / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
double strand RNA binding domain from DEAD END PROTEIN 1 / Ribonuclease III / Dicer dimerisation domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family ...double strand RNA binding domain from DEAD END PROTEIN 1 / Ribonuclease III / Dicer dimerisation domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer double-stranded RNA-binding fold 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. / Double-stranded RNA binding motif / Helicase/UvrB, N-terminal / Type III restriction enzyme, res subunit / Double-stranded RNA binding motif / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / Helicase conserved C-terminal domain / helicase superfamily 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
Endoribonuclease Dicer homolog 1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsWei X / Ke H / Feng Y
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31970040 China
CitationJournal: Nat Plants / Year: 2021
Title: Structural basis of microRNA processing by Dicer-like 1.
Authors: Xiaobin Wei / Huanhuan Ke / Aijia Wen / Bo Gao / Jing Shi / Yu Feng /
Abstract: MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. In animals, pri-miRNAs are cleaved by Drosha to generate pre-miRNAs, which ...MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. In animals, pri-miRNAs are cleaved by Drosha to generate pre-miRNAs, which are subsequently cleaved by Dicer to generate mature miRNAs. Instead of being cleaved by two different enzymes, both cleavages in plants are performed by Dicer-like 1 (DCL1). With a similar domain architecture as human Dicer, it is mysterious how DCL1 recognizes pri-miRNAs and performs two cleavages sequentially. Here, we report the single-particle cryo-electron microscopy structures of Arabidopsis DCL1 complexed with a pri-miRNA and a pre-miRNA, respectively, in cleavage-competent states. These structures uncover the plasticity of the PAZ domain, which is critical for the recognition of both pri-miRNA and pre-miRNA. These structures suggest that the helicase module serves as an engine that transfers the substrate between two sequential cleavage events. This study lays a foundation for dissecting the regulation mechanism of miRNA biogenesis in plants and provides insights into the dicing state of human Dicer.
History
DepositionApr 9, 2021-
Header (metadata) releaseAug 11, 2021-
Map releaseAug 11, 2021-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ele
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31182.png

Downloads & links

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Map

FileDownload / File: emd_31182.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.014 Å
Density
Contour LevelBy AUTHOR: 0.006 / Movie #1: 0.006
Minimum - Maximum-0.011615287 - 0.027873216
Average (Standard dev.)1.3891849e-05 (±0.0016969546)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 202.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0141.0141.014
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z202.800202.800202.800
α/β/γ90.00090.00090.000
start NX/NY/NZ594743
NX/NY/NZ375263
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0120.0280.000

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Supplemental data

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Sample components

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Entire : Binary complex of DCL1 in complex with pre-miRNA 166f

EntireName: Binary complex of DCL1 in complex with pre-miRNA 166f
Components
  • Complex: Binary complex of DCL1 in complex with pre-miRNA 166f
    • Protein or peptide: Endoribonuclease Dicer homolog 1
    • RNA: pre-miRNA 166f

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Supramolecule #1: Binary complex of DCL1 in complex with pre-miRNA 166f

SupramoleculeName: Binary complex of DCL1 in complex with pre-miRNA 166f / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Arabidopsis thaliana (thale cress)

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Macromolecule #1: Endoribonuclease Dicer homolog 1

MacromoleculeName: Endoribonuclease Dicer homolog 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 213.859344 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MVMEDEPREA TIKPSYWLDA CEDISCDLID DLVSEFDPSS VAVNESTDEN GVINDFFGGI DHILDSIKNG GGLPNNGVSD TNSQINEVT VTPQVIAKET VKENGLQKNG GKRDEFSKEE GDKDRKRARV CSYQSERSNL SGRGHVNNSR EGDRFMNRKR T RNWDEAGN ...String:
MVMEDEPREA TIKPSYWLDA CEDISCDLID DLVSEFDPSS VAVNESTDEN GVINDFFGGI DHILDSIKNG GGLPNNGVSD TNSQINEVT VTPQVIAKET VKENGLQKNG GKRDEFSKEE GDKDRKRARV CSYQSERSNL SGRGHVNNSR EGDRFMNRKR T RNWDEAGN NKKKRECNNY RRDGRDREVR GYWERDKVGS NELVYRSGTW EADHERDVKK VSGGNRECDV KAEENKSKPE ER KEKVVEE QARRYQLDVL EQAKAKNTIA FLETGAGKTL IAILLIKSVH KDLMSQNRKM LSVFLVPKVP LVYQQAEVIR NQT CFQVGH YCGEMGQDFW DSRRWQREFE SKQVLVMTAQ ILLNILRHSI IRMETIDLLI LDECHHAVKK HPYSLVMSEF YHTT PKDKR PAIFGMTASP VNLKGVSSQV DCAIKIRNLE TKLDSTVCTI KDRKELEKHV PMPSEIVVEY DKAATMWSLH ETIKQ MIAA VEEAAQASSR KSKWQFMGAR DAGAKDELRQ VYGVSERTES DGAANLIHKL RAINYTLAEL GQWCAYKVGQ SFLSAL QSD ERVNFQVDVK FQESYLSEVV SLLQCELLEG AAAEKVAAEV GKPENGNAHD EMEEGELPDD PVVSGGEHVD EVIGAAV AD GKVTPKVQSL IKLLLKYQHT ADFRAIVFVE RVVAALVLPK VFAELPSLSF IRCASMIGHN NSQEMKSSQM QDTISKFR D GHVTLLVATS VAEEGLDIRQ CNVVMRFDLA KTVLAYIQSR GRARKPGSDY ILMVERGNVS HAAFLRNARN SEETLRKEA IERTDLSHLK DTSRLISIDA VPGTVYKVEA TGAMVSLNSA VGLVHFYCSQ LPGDRYAILR PEFSMEKHEK PGGHTEYSCR LQLPCNAPF EILEGPVCSS MRLAQQAVCL AACKKLHEMG AFTDMLLPDK GSGQDAEKAD QDDEGEPVPG TARHREFYPE G VADVLKGE WVSSGKEVCE SSKLFHLYMY NVRCVDFGSS KDPFLSEVSE FAILFGNELD AEVLSMSMDL YVARAMITKA SL AFKGSLD ITENQLSSLK KFHVRLMSIV LDVDVEPSTT PWDPAKAYLF VPVTDNTSME PIKGINWELV EKITKTTAWD NPL QRARPD VYLGTNERTL GGDRREYGFG KLRHNIVFGQ KSHPTYGIRG AVASFDVVRA SGLLPVRDAF EKEVEEDLSK GKLM MADGC MVAEDLIGKI VTAAHSGKRF YVDSICYDMS AETSFPRKEG YLGPLEYNTY ADYYKQKYGV DLNCKQQPLI KGRGV SYCK NLLSPRFEQS GESETVLDKT YYVFLPPELC VVHPLSGSLI RGAQRLPSIM RRVESMLLAV QLKNLISYPI PTSKIL EAL TAASCQETFC YERAELLGDA YLKWVVSRFL FLKYPQKHEG QLTRMRQQMV SNMVLYQFAL VKGLQSYIQA DRFAPSR WS APGVPPVFDE DTKDGGSSFF DEEQKPVSEE NSDVFEDGEM EDGELEGDLS SYRVLSSKTL ADVVEALIGV YYVEGGKI A ANHLMKWIGI HVEDDPDEVD GTLKNVNVPE SVLKSIDFVG LERALKYEFK EKGLLVEAIT HASRPSSGVS CYQRLEFVG DAVLDHLITR HLFFTYTSLP PGRLTDLRAA AVNNENFARV AVKHKLHLYL RHGSSALEKQ IREFVKEVQT ESSKPGFNSF GLGDCKAPK VLGDIVESIA GAIFLDSGKD TTAAWKVFQP LLQPMVTPET LPMHPVRELQ ERCQQQAEGL EYKASRSGNT A TVEVFIDG VQVGVAQNPQ KKMAQKLAAR NALAALKEKE IAESKEKHIN NGNAGEDQGE NENGNKKNGH QPFTRQTLND IC LRKNWPM PSYRCVKEGG PAHAKRFTFG VRVNTSDRGW TDECIGEPMP SVKKAKDSAA VLLLELLNKT FS

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Macromolecule #2: pre-miRNA 166f

MacromoleculeName: pre-miRNA 166f / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Arabidopsis thaliana (thale cress)
Molecular weightTheoretical: 28.454812 KDa
SequenceString:
UGAAUGAUGC CUGGCUCGAG ACCAUUCAAU CUCAUGAUCU CAUGAUUAUA ACGAUGAUGA UGAUGAUGUC GGACCAGGCU UCAUUCCCC

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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

BufferpH: 7.5
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: 64.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 300148

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