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- EMDB-30005: pri-miRNA bound DROSHA-DGCR8 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-30005
Titlepri-miRNA bound DROSHA-DGCR8 complex
Map data
Sample
  • Complex: Ternary complex of DROSHA-DGCR8 with pri-16-1m RNA
    • Protein or peptide: Ribonuclease 3
    • Protein or peptide: Microprocessor complex subunit DGCR8
    • RNA: RNA (102-mer)
  • Ligand: ZINC ION
KeywordsRibonuclease / RNA BINDING PROTEIN / HYDROLASE-RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


positive regulation of pre-miRNA processing / protein-RNA adaptor activity / regulation of miRNA metabolic process / DEAD/H-box RNA helicase binding / primary miRNA binding / regulation of regulatory T cell differentiation / Transcriptional Regulation by MECP2 / ribonuclease III / miRNA metabolic process / primary miRNA processing ...positive regulation of pre-miRNA processing / protein-RNA adaptor activity / regulation of miRNA metabolic process / DEAD/H-box RNA helicase binding / primary miRNA binding / regulation of regulatory T cell differentiation / Transcriptional Regulation by MECP2 / ribonuclease III / miRNA metabolic process / primary miRNA processing / regulation of stem cell proliferation / microprocessor complex / pre-miRNA processing / ribonuclease III activity / MicroRNA (miRNA) biogenesis / SMAD binding / R-SMAD binding / lipopolysaccharide binding / rRNA processing / double-stranded RNA binding / regulation of inflammatory response / defense response to Gram-negative bacterium / postsynaptic density / nuclear body / defense response to Gram-positive bacterium / glutamatergic synapse / heme binding / positive regulation of gene expression / nucleolus / protein homodimerization activity / RNA binding / nucleoplasm / identical protein binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
RNase III, double-stranded RNA binding domain, animal / Microprocessor complex subunit DGCR8 / Ribonuclease-III-like / Ribonuclease III / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / Double-stranded RNA binding motif ...RNase III, double-stranded RNA binding domain, animal / Microprocessor complex subunit DGCR8 / Ribonuclease-III-like / Ribonuclease III / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / Double-stranded RNA binding motif / Double-stranded RNA binding motif / Ribonuclease III, endonuclease domain superfamily / Double stranded RNA-binding domain (dsRBD) profile. / Double-stranded RNA-binding domain / WW domain superfamily / WW/rsp5/WWP domain profile. / Domain with 2 conserved Trp (W) residues / WW domain
Similarity search - Domain/homology
Microprocessor complex subunit DGCR8 / Ribonuclease 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsJin W / Wang J
Funding support China, 6 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31521002 China
National Natural Science Foundation of China (NSFC)31700721 China
National Natural Science Foundation of China (NSFC)31825009 China
Ministry of Science and Technology (MoST, China)2016YFA0501100 China
Beijing Municipal Science and Technology ProjectZ171100000417001 China
Chinese Academy of Sciences2018125 China
CitationJournal: Mol Cell / Year: 2020
Title: Structural Basis for pri-miRNA Recognition by Drosha.
Authors: Wenxing Jin / Jia Wang / Chao-Pei Liu / Hong-Wei Wang / Rui-Ming Xu /
Abstract: A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the ...A commencing and critical step in miRNA biogenesis involves processing of pri-miRNAs in the nucleus by Microprocessor. An important, but not completely understood, question is how Drosha, the catalytic subunit of Microprocessor, binds pri-miRNAs and correctly specifies cleavage sites. Here we report the cryoelectron microscopy structures of the Drosha-DGCR8 complex with and without a pri-miRNA. The RNA-bound structure provides direct visualization of the tertiary structure of pri-miRNA and shows that a helix hairpin in the extended PAZ domain and the mobile basic (MB) helix in the RNase IIIa domain of Drosha coordinate to recognize the single-stranded to double-stranded junction of RNA, whereas the dsRNA binding domain makes extensive contacts with the RNA stem. Furthermore, the RNA-free structure reveals an autoinhibitory conformation of the PAZ helix hairpin. These findings provide mechanistic insights into pri-miRNA cleavage site selection and conformational dynamics governing pri-miRNA recognition by the catalytic component of Microprocessor.
History
DepositionFeb 10, 2020-
Header (metadata) releaseApr 15, 2020-
Map releaseApr 15, 2020-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6lxd
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30005.png

Downloads & links

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Map

FileDownload / File: emd_30005.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.029856745 - 0.06778992
Average (Standard dev.)0.00023928887 (±0.002438278)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 243.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z243.000243.000243.000
α/β/γ90.00090.00090.000
start NX/NY/NZ636181
NX/NY/NZ13113592
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0300.0680.000

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Supplemental data

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Sample components

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Entire : Ternary complex of DROSHA-DGCR8 with pri-16-1m RNA

EntireName: Ternary complex of DROSHA-DGCR8 with pri-16-1m RNA
Components
  • Complex: Ternary complex of DROSHA-DGCR8 with pri-16-1m RNA
    • Protein or peptide: Ribonuclease 3
    • Protein or peptide: Microprocessor complex subunit DGCR8
    • RNA: RNA (102-mer)
  • Ligand: ZINC ION

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Supramolecule #1: Ternary complex of DROSHA-DGCR8 with pri-16-1m RNA

SupramoleculeName: Ternary complex of DROSHA-DGCR8 with pri-16-1m RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Ribonuclease 3

MacromoleculeName: Ribonuclease 3 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ribonuclease III
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 115.390578 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EPEETMPDKN EEEEEELLKP VWIRCTHSEN YYSSDPMDQV GDSTVVGTSR LRDLYDKFEE ELGSRQEKAK AARPPWEPPK TKLDEDLES SSESECESDE DSTCSSSSDS EVFDVIAEIK RKKAHPDRLH DELWYNDPGQ MNDGPLCKCS AKARRTGIRH S IYPGEEAI ...String:
EPEETMPDKN EEEEEELLKP VWIRCTHSEN YYSSDPMDQV GDSTVVGTSR LRDLYDKFEE ELGSRQEKAK AARPPWEPPK TKLDEDLES SSESECESDE DSTCSSSSDS EVFDVIAEIK RKKAHPDRLH DELWYNDPGQ MNDGPLCKCS AKARRTGIRH S IYPGEEAI KPCRPMTNNA GRLFHYRITV SPPTNFLTDR PTVIEYDDHE YIFEGFSMFA HAPLTNIPLC KVIRFNIDYT IH FIEEMMP ENFCVKGLEL FSLFLFRDIL ELYDWNLKGP LFEDSPPCCP RFHFMPRFVR FLPDGGKEVL SMHQILLYLL RCS KALVPE EEIANMLQWE ELEWQKYAEE CKGMIVTNPG TKPSSVRIDQ LDREQFNPDV ITFPIIVHFG IRPAQLSYAG DPQY QKLWK SYVKLRHLLA NSPKVKQTDK QKLAQREEAL QKIRQKNTMR REVTVELSSQ GFWKTGIRSD VCQHAMMLPV LTHHI RYHQ CLMHLDKLIG YTFQDRCLLQ LAMTHPSHHL NFGMNPDHAR NSLSNCGIRQ PKYGDRKVHH MHMRKKGINT LINIMS RLG QDDPTPSRIN HNERLEFLGD AVVEFLTSVH LYYLFPSLEE GGLATYRTAI VQNQHLAMLA KKLELDRFML YAHGPDL CR ESDLRHAMAN CFQALIGAVY LEGSLEEAKQ LFGRLLFNDP DLREVWLNYP LHPLQLQEPN TDRQLIETSP VLQKLTEF E EAIGVIFTHV RLLARAFTLR TVGFNHLTLG HNQRMEFLGD SIMQLVATEY LFIHFPDHHE GHLTLLRSSL VNNRTQAKV AEELGMQEYA ITNDKTKRPV ALRTKTLADL LQSFIAALYI DKDLEYVHTF MNVCFFPRLK EFILNQDWND PKSQLQQCCL TLRTEGKEP DIPLYKTLQT VGPSHARTYT VAVYFKGERI GCGKGPSIQQ AEMGAAMDAL EKYNFPQMAH QKRFIERKYR Q ELKEMRWE REHQEREPDE TEDIKKHHHH HH

UniProtKB: Ribonuclease 3

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Macromolecule #2: Microprocessor complex subunit DGCR8

MacromoleculeName: Microprocessor complex subunit DGCR8 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 86.171203 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: METDESPSPL PCGPAGEAVM ESRARPFQAL PREQSPPPPL QTSSGAEVMD VGSGGDGQSE LPAEDPFNFY GASLLSKGSF SKGRLLIDP NCSGHSPRTA RHAPAVRKFS PDLKLLKDVK ISVSFTESCR SKDRKVLYTG AERDVRAECG LLLSPVSGDV H ACPFGGSV ...String:
METDESPSPL PCGPAGEAVM ESRARPFQAL PREQSPPPPL QTSSGAEVMD VGSGGDGQSE LPAEDPFNFY GASLLSKGSF SKGRLLIDP NCSGHSPRTA RHAPAVRKFS PDLKLLKDVK ISVSFTESCR SKDRKVLYTG AERDVRAECG LLLSPVSGDV H ACPFGGSV GDGVGIGGES ADKKDEENEL DQEKRVEYAV LDELEDFTDN LELDEEGAGG FTAKAIVQRD RVDEEALNFP YE DDFDNDV DALLEEGLCA PKKRRTEEKY GGDSDHPSDG ETSVQPMMTK IKTVLKSRGR PPTEPLPDGW IMTFHNSGVP VYL HRESRV VTWSRPYFLG TGSIRKHDPP LSSIPCLHYK KMKDNEEREQ SSDLTPSGDV SPVKPLSRSA ELEFPLDEPD SMGA DPGPP DEKDPLGAEA APGALGQVKA KVEVCKDESV DLEEFRSYLE KRFDFEQVTV KKFRTWAERR QFNREMKRKQ AESER PILP ANQKLITLSV QDAPTKKEFV INPNGKSEVC ILHEYMQRVL KVRPVYNFFE CENPSEPFGA SVTIDGVTYG SGTASS KKL AKNKAARATL EILIPDFVKQ TSEEKPKDSE ELEYFNHISI EDSRVYELTS KAGLLSPYQI LHECLKRNHG MGDTSIK FE VVPGKNQKSE YVMACGKHTV RGWCKNKRVG KQLASQKILQ LLHPHVKNWG SLLRMYGRES SKMVKQETSD KSVIELQQ Y AKKNKPNLHI LSKLQEEMKR LAEEREETRK KPKMSIVASA QPGGEPLCTV DV

UniProtKB: Microprocessor complex subunit DGCR8

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Macromolecule #3: RNA (102-mer)

MacromoleculeName: RNA (102-mer) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 32.759398 KDa
SequenceString:
GGUGAUAGCA AUGUCAGCAG UGCCUUAGCA GCACGUAAAU AUUGGCCAUU GCACUCCGGC CAGUAUUAAC UGUGCUGCUG AAGUAAGGU UGACCAUACU CUA

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / 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

Concentration0.3 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / 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 opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
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

Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 250943

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Atomic model buiding 1

Initial modelPDB ID:

5b16


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-6lxd:
Pri-miRNA bound DROSHA-DGCR8 complex

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