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- EMDB-21052: Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA c... -

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Basic information

Entry
Database: EMDB / ID: EMD-21052
TitleHuman Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
Map dataHuman Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
Sample
  • Complex: Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
    • Protein or peptide: Ribonuclease 3
    • Protein or peptide: Microprocessor complex subunit DGCR8
    • RNA: Pri-miR-16-2 (66-MER)
KeywordsMicroprocessor / Drosha / DGCR8 / Primary MicroRNA / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


positive regulation of pre-miRNA processing / regulation of miRNA metabolic process / protein-RNA adaptor activity / DEAD/H-box RNA helicase binding / regulation of regulatory T cell differentiation / primary miRNA binding / Transcriptional Regulation by MECP2 / ribonuclease III / miRNA metabolic process / primary miRNA processing ...positive regulation of pre-miRNA processing / regulation of miRNA metabolic process / protein-RNA adaptor activity / DEAD/H-box RNA helicase binding / regulation of regulatory T cell differentiation / primary miRNA binding / Transcriptional Regulation by MECP2 / ribonuclease III / miRNA metabolic process / primary miRNA processing / regulation of stem cell proliferation / ribonuclease III activity / microprocessor complex / pre-miRNA processing / MicroRNA (miRNA) biogenesis / SMAD binding / R-SMAD binding / lipopolysaccharide binding / rRNA processing / double-stranded RNA binding / site of double-strand break / protein-macromolecule adaptor activity / regulation of inflammatory response / defense response to Gram-negative bacterium / nuclear body / postsynaptic density / defense response to Gram-positive bacterium / heme binding / DNA damage response / positive regulation of gene expression / nucleolus / glutamatergic synapse / protein homodimerization activity / RNA binding / nucleoplasm / identical protein binding / nucleus / metal ion binding / 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: 4.4 Å
AuthorsPartin A / Zhang K
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM122960 United States
CitationJournal: Mol Cell / Year: 2020
Title: Cryo-EM Structures of Human Drosha and DGCR8 in Complex with Primary MicroRNA.
Authors: Alexander C Partin / Kaiming Zhang / Byung-Cheon Jeong / Emily Herrell / Shanshan Li / Wah Chiu / Yunsun Nam /
Abstract: Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding ...Metazoan microRNAs require specific maturation steps initiated by Microprocessor, comprising Drosha and DGCR8. Lack of structural information for the assembled complex has hindered an understanding of how Microprocessor recognizes primary microRNA transcripts (pri-miRNAs). Here we present a cryoelectron microscopy structure of human Microprocessor with a pri-miRNA docked in the active site, poised for cleavage. The basal junction is recognized by a four-way intramolecular junction in Drosha, triggered by the Belt and Wedge regions that clamp over the ssRNA. The belt is important for efficiency and accuracy of pri-miRNA processing. Two dsRBDs form a molecular ruler to measure the stem length between the two dsRNA-ssRNA junctions. The specific organization of the dsRBDs near the apical junction is independent of Drosha core domains, as observed in a second structure in the partially docked state. Collectively, we derive a molecular model to explain how Microprocessor recognizes a pri-miRNA and accurately identifies the cleavage site.
History
DepositionDec 4, 2019-
Header (metadata) releaseApr 8, 2020-
Map releaseApr 8, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v5c
  • Surface level: 0.6
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21052.png

Downloads & links

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Map

FileDownload / File: emd_21052.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHuman Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å		1.06 Å/pix.
x 256 pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6 / Movie #1: 0.6
Minimum - Maximum-1.1694232 - 2.361421
Average (Standard dev.)0.0074751033 (±0.09056183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.360271.360271.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.1692.3610.007

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Supplemental data

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Sample components

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Entire : Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA c...

EntireName: Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
Components
  • Complex: Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
    • Protein or peptide: Ribonuclease 3
    • Protein or peptide: Microprocessor complex subunit DGCR8
    • RNA: Pri-miR-16-2 (66-MER)

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Supramolecule #1: Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA c...

SupramoleculeName: Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40 KDa

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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: 118.377742 KDa
Recombinant expressionOrganism: Baculovirus expression vector pFastBac1-HM
SequenceString: GSGHRSPSRE KKRARWEEEK DRWSDNQSSG KDKNYTSIKE KEPEETMPDK NEEEEEELLK PVWIRCTHSE NYYSSDPMDQ VGDSTVVGT SRLRDLYDKF EEELGSRQEK AKAARPPWEP PKTKLDEDLE SSSESECESD EDSTCSSSSD SEVFDVIAEI K RKKAHPDR ...String:
GSGHRSPSRE KKRARWEEEK DRWSDNQSSG KDKNYTSIKE KEPEETMPDK NEEEEEELLK PVWIRCTHSE NYYSSDPMDQ VGDSTVVGT SRLRDLYDKF EEELGSRQEK AKAARPPWEP PKTKLDEDLE SSSESECESD EDSTCSSSSD SEVFDVIAEI K RKKAHPDR LHDELWYNDP GQMNDGPLCK CSAKARRTGI RHSIYPGEEA IKPCRPMTNN AGRLFHYRIT VSPPTNFLTD RP TVIEYDD HEYIFEGFSM FAHAPLTNIP LCKVIRFNID YTIHFIEEMM PENFCVKGLE LFSLFLFRDI LELYDWNLKG PLF EDSPPC CPRFHFMPRF VRFLPDGGKE VLSMHQILLY LLRCSKALVP EEEIANMLQW EELEWQKYAE ECKGMIVTNP GTKP SSVRI DQLDREQFNP DVITFPIIVH FGIRPAQLSY AGDPQYQKLW KSYVKLRHLL ANSPKVKQTD KQKLAQREEA LQKIR QKNT MRREVTVELS SQGFWKTGIR SDVCQHAMML PVLTHHIRYH QCLMHLDKLI GYTFQDRCLL QLAMTHPSHH LNFGMN PDH ARNSLSNCGI RQPKYGDRKV HHMHMRKKGI NTLINIMSRL GQDDPTPSRI NHNERLEFLG DAVVEFLTSV HLYYLFP SL EEGGLATYRT AIVQNQHLAM LAKKLELDRF MLYAHGPDLC RESDLRHAMA NCFQALIGAV YLEGSLEEAK QLFGRLLF N DPDLREVWLN YPLHPLQLQE PNTDRQLIET SPVLQKLTEF EEAIGVIFTH VRLLARAFTL RTVGFNHLTL GHNQRMEFL GDSIMQLVAT EYLFIHFPDH HEGHLTLLRS SLVNNRTQAK VAEELGMQEY AITNDKTKRP VALRTKTLAD LLQSFIAALY IDKDLEYVH TFMNVCFFPR LKEFILNQDW NDPKSQLQQC CLTLRTEGKE PDIPLYKTLQ TVGPSHARTY TVAVYFKGER I GCGKGPSI QQAEMGAAMD ALEKYNFPQM AHQKRFIERK YRQELKEMRW EREHQERE

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: 60.550602 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: GSGAIVQRDR VDEEALNFPY EDDFDNDVDA LLEEGLCAPK KRRTEEKYGG DSDHPSDGET SVQPMMTKIK TVLKSRGRPP TEPLPDGWI MTFHNSGVPV YLHRESRVVT WSRPYFLGTG SIRKHDPPLS SIPCLHYKKM KDNEEREQSS DLTPSGDVSP V KPLSRSAE ...String:
GSGAIVQRDR VDEEALNFPY EDDFDNDVDA LLEEGLCAPK KRRTEEKYGG DSDHPSDGET SVQPMMTKIK TVLKSRGRPP TEPLPDGWI MTFHNSGVPV YLHRESRVVT WSRPYFLGTG SIRKHDPPLS SIPCLHYKKM KDNEEREQSS DLTPSGDVSP V KPLSRSAE LEFPLDEPDS MGADPGPPDE KDPLGAEAAP GALGQVKAKV EVCKDESVDL EEFRSYLEKR FDFEQVTVKK FR TWAERRQ FNREMKRKQA ESERPILPAN QKLITLSVQD APTKKEFVIN PNGKSEVCIL HEYMQRVLKV RPVYNFFECE NPS EPFGAS VTIDGVTYGS GTASSKKLAK NKAARATLEI LIPDFVKQTS EEKPKDSEEL EYFNHISIED SRVYELTSKA GLLS PYQIL HECLKRNHGM GDTSIKFEVV PGKNQKSEYV MACGKHTVRG WCKNKRVGKQ LASQKILQLL HPHVKNWGSL LRMYG RESS KMVKQETSDK SVIELQQYAK KNKPNLHILS KLQEEMKRLA EEREETRK

UniProtKB: Microprocessor complex subunit DGCR8

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Macromolecule #3: Pri-miR-16-2 (66-MER)

MacromoleculeName: Pri-miR-16-2 (66-MER) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 33.646934 KDa
SequenceString:
CUGACAUACU UGUUCCACUC UAGCAGCACG UAAAUAUUGG CGUAGUGAAA UAUAUAUUAA ACACCAAUAU UACUGUGCUG CUUUAGUGU GACAGGGAUA CAGCAA

GENBANK: GENBANK: AC024221.23

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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

Concentration3.7 mg/mL
BufferpH: 7.1
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsHuman Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-30 / Number grids imaged: 4 / Number real images: 6070 / Average exposure time: 6.0 sec. / Average electron dose: 46.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
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: 1063710
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.0.5) / Number images used: 381468
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.2)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.0.2)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: BACKBONE TRACE
Output model

PDB-6v5c:
Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state

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