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- EMDB-43821: Human Drosha and DGCR8 in complex with Pri-let-7a2 -

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Basic information

Entry
Database: EMDB / ID: EMD-43821
TitleHuman Drosha and DGCR8 in complex with Pri-let-7a2
Map data
Sample
  • Complex: RNAi_protein_a2
    • Protein or peptide: Isoform 4 of Drosha
    • Protein or peptide: Microprocessor complex subunit DGCR8
    • RNA: Pri-let-7a2
  • Ligand: ZINC ION
  • Ligand: CALCIUM ION
  • Ligand: water
KeywordsRNAi / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


positive regulation of pre-miRNA processing / microprocessor complex / regulation of miRNA metabolic process / protein-RNA adaptor activity / DEAD/H-box RNA helicase binding / primary miRNA binding / ribonuclease III / Transcriptional Regulation by MECP2 / regulation of regulatory T cell differentiation / primary miRNA processing ...positive regulation of pre-miRNA processing / microprocessor complex / regulation of miRNA metabolic process / protein-RNA adaptor activity / DEAD/H-box RNA helicase binding / primary miRNA binding / ribonuclease III / Transcriptional Regulation by MECP2 / regulation of regulatory T cell differentiation / primary miRNA processing / miRNA metabolic process / regulation of stem cell proliferation / ribonuclease III activity / pre-miRNA processing / MicroRNA (miRNA) biogenesis / SMAD binding / R-SMAD binding / lipopolysaccharide binding / rRNA processing / double-stranded RNA binding / site of double-strand break / regulation of inflammatory response / protein-macromolecule adaptor activity / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium / postsynaptic density / nuclear body / heme binding / DNA damage response / positive regulation of gene expression / nucleolus / glutamatergic synapse / protein homodimerization activity / RNA binding / nucleoplasm / metal ion binding / identical protein binding / nucleus / cytoplasm / cytosol
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: 2.9 Å
AuthorsGarg A / Joshua-Tor L
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Cancer Institute (NIH/NCI)R01 GM114147 United States
CitationJournal: Mol Cell / Year: 2024
Title: The structural landscape of Microprocessor-mediated processing of pri-let-7 miRNAs.
Authors: Ankur Garg / Renfu Shang / Todor Cvetanovic / Eric C Lai / Leemor Joshua-Tor /
Abstract: MicroRNA (miRNA) biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri- ...MicroRNA (miRNA) biogenesis is initiated upon cleavage of a primary miRNA (pri-miRNA) hairpin by the Microprocessor (MP), composed of the Drosha RNase III enzyme and its partner DGCR8. Multiple pri-miRNA sequence motifs affect MP recognition, fidelity, and efficiency. Here, we performed cryoelectron microscopy (cryo-EM) and biochemical studies of several let-7 family pri-miRNAs in complex with human MP. We show that MP has the structural plasticity to accommodate a range of pri-miRNAs. These structures revealed key features of the 5' UG sequence motif, more comprehensively represented as the "flipped U with paired N" (fUN) motif. Our analysis explains how cleavage of class-II pri-let-7 members harboring a bulged nucleotide generates a non-canonical precursor with a 1-nt 3' overhang. Finally, the MP-SRSF3-pri-let-7f1 structure reveals how SRSF3 contributes to MP fidelity by interacting with the CNNC motif and Drosha's Piwi/Argonaute/Zwille (PAZ)-like domain. Overall, this study sheds light on the mechanisms for flexible recognition, accurate cleavage, and regulated processing of different pri-miRNAs by MP.
History
DepositionFeb 26, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43821.png

Downloads & links

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Map

FileDownload / File: emd_43821.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 440 pix.
= 376.64 Å		0.86 Å/pix.
x 440 pix.
= 376.64 Å		0.86 Å/pix.
x 440 pix.
= 376.64 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.856 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.08
Minimum - Maximum-0.1626159 - 0.6046471
Average (Standard dev.)-0.000004849446 (±0.01023843)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 376.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43821_msk_1.map
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Half map: #2

Fileemd_43821_half_map_1.map
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Half map: #1

Fileemd_43821_half_map_2.map
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Sample components

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Entire : RNAi_protein_a2

EntireName: RNAi_protein_a2
Components
  • Complex: RNAi_protein_a2
    • Protein or peptide: Isoform 4 of Drosha
    • Protein or peptide: Microprocessor complex subunit DGCR8
    • RNA: Pri-let-7a2
  • Ligand: ZINC ION
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: RNAi_protein_a2

SupramoleculeName: RNAi_protein_a2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: Isoform 4 of Drosha

MacromoleculeName: Isoform 4 of Drosha / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: ribonuclease III
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 155.574297 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MMQGNTCHRM SFHPGRGCPR GRGGHGARPS APSFRPQNLR LLHPQQPPVQ YQYEPPSAPS TTFSNSPAPN FLPPRPDFVP FPPPMPPSA QGPLPPCPIR PPFPNHQMRH PFPVPPCFPP MPPPMPCPNN PPVPGAPPGQ GTFPFMMPPP SMPHPPPPPV M PQQVNYQY ...String:
MMQGNTCHRM SFHPGRGCPR GRGGHGARPS APSFRPQNLR LLHPQQPPVQ YQYEPPSAPS TTFSNSPAPN FLPPRPDFVP FPPPMPPSA QGPLPPCPIR PPFPNHQMRH PFPVPPCFPP MPPPMPCPNN PPVPGAPPGQ GTFPFMMPPP SMPHPPPPPV M PQQVNYQY PPGYSHHNFP PPSFNSFQNN PSSFLPSANN SSSPHFRHLP PYPLPKAPSE RRSPERLKHY DDHRHRDHSH GR GERHRSL DRRERGRSPD RRRQDSRYRS DYDRGRTPSR HRSYERSRER ERERHRHRDN RRSPSLERSY KKEYKRSGSR SPS REKKRA RWEEEKDRWS DNQSSGKDKN YTSIKEKEPE ETMPDKNEEE EEELLKPVWI RCTHSENYYS SDPMDQVGDS TVVG TSRLR DLYDKFEEEL GSRQEKAKAA RPPWEPPKTK LDEDLESSSE SECESDEDST CSSSSDSEVF DVIAEIKRKK AHPDR LHDE LWYNDPGQMN DGPLCKCSAK ARRTGIRHSI YPGEEAIKPC RPMTNNAGRL FHYRITVSPP TNFLTDRPTV IEYDDH EYI FEGFSMFAHA PLTNIPLCKV IRFNIDYTIH FIEEMMPENF CVKGLELFSL FLFRDILELY DWNLKGPLFE DSPPCCP RF HFMPRFVRFL PDGGKEVLSM HQILLYLLRC SKALVPEEEI ANMLQWEELE WQKYAEECKG MIVTNPGTKP SSVRIDQL D REQFNPDVIT FPIIVHFGIR PAQLSYAGDP QYQKLWKSYV KLRHLLANSP KVKQTDKQKL AQREEALQKI RQKNTMRRE VTVELSSQGF WKTGIRSDVC QHAMMLPVLT HHIRYHQCLM HLDKLIGYTF QDRCLLQLAM THPSHHLNFG MNPDHARNSL SNCGIRQPK YGDRKVHHMH MRKKGINTLI NIMSRLGQDD PTPSRINHNE RLEFLGDAVV EFLTSVHLYY LFPSLEEGGL A TYRTAIVQ NQHLAMLAKK LELDRFMLYA HGPDLCRESD LRHAMANCFE ALIGAVYLEG SLEEAKQLFG RLLFNDPDLR EV WLNYPLH PLQLQEPNTD RQLIETSPVL QKLTEFEEAI GVIFTHVRLL ARAFTLRTVG FNHLTLGHNQ RMEFLGDSIM QLV ATEYLF IHFPDHHEGH LTLLRSSLVN NRTQAKVAEE LGMQEYAITN DKTKRPVALR TKTLADLLES FIAALYIDKD LEYV HTFMN VCFFPRLKEF ILNQDWNDPK SQLQQCCLTL RTEGKEPDIP LYKTLQTVGP SHARTYTVAV YFKGERIGCG KGPSI QQAE MGAAMDALEK YNFPQMAHQK RFIERKYRQE LKEMRWEREH QEREPDETED IKK

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: Pri-let-7a2

MacromoleculeName: Pri-let-7a2 / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.783082 KDa
SequenceString:
UCCAGCCAUU GUGACUGCAU GCUCCCAGGU UGAGGUAGUA GGUUGUAUAG UUUAGAAUUA CAUCAAGGGA GAUAACUGUA CAGCCUCCU AGCUUUCCUU GGGUCUUGCA CUAAACAACA UGGUGAGA

GENBANK: GENBANK: AP001359.4

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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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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 2 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 78.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.6 µm
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

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 251413
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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