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- PDB-6v5c: Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA c... -

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Basic information

Entry
Database: PDB / ID: 6v5c
TitleHuman Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
Components
  • Microprocessor complex subunit DGCR8
  • Pri-miR-16-2 (66-MER)
  • Ribonuclease 3
KeywordsRNA BINDING PROTEIN/RNA / Microprocessor / 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 / postsynaptic density / nuclear body / defense response to Gram-positive bacterium / glutamatergic synapse / heme binding / DNA damage response / positive regulation of gene expression / nucleolus / 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
: / RNA / RNA (> 10) / RNA (> 100) / Microprocessor complex subunit DGCR8 / Ribonuclease 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsPartin, A. / Zhang, K. / Jeong, B. / Herrell, E. / Li, S. / Chiu, W. / Nam, Y.
Funding support United States, 4items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 8, 2020Provider: repository / Type: Initial release
Revision 1.1May 20, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2May 27, 2020Group: Database references / Category: citation / Item: _citation.page_last
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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  • Deposited structure unit
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  • EMDB-21052
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Assembly

Deposited unit
A: Ribonuclease 3
C: Microprocessor complex subunit DGCR8
B: Microprocessor complex subunit DGCR8
D: Pri-miR-16-2 (66-MER)


Theoretical massNumber of molelcules
Total (without water)273,1264
Polymers273,1264
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Ribonuclease 3 / Protein Drosha / Ribonuclease III / RNase III / p241


Mass: 118377.742 Da / Num. of mol.: 1 / Mutation: E1045Q, E1222Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DROSHA, RN3, RNASE3L, RNASEN / Production host: Baculovirus expression vector pFastBac1-HM / References: UniProt: Q9NRR4, ribonuclease III
#2: Protein Microprocessor complex subunit DGCR8 / DiGeorge syndrome critical region 8


Mass: 60550.602 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DGCR8, C22orf12, DGCRK6, LP4941
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q8WYQ5
#3: RNA chain Pri-miR-16-2 (66-MER)


Mass: 33646.934 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Transcription vector pUC18-pES2 (others) / References: GenBank: 21206011

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.35 MDaYES
210.040 MDaYES
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.1
SpecimenConc.: 3.7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Human Drosha and DGCR8 in complex with Primary MicroRNA (MP/RNA complex) - partially docked state
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3600 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 46.8 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 4 / Num. of real images: 6070
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
7UCSF Chimera1.13.1model fitting
9PHENIX1.14model refinement
10RELION3.0.2initial Euler assignment
11RELION3.0.2final Euler assignment
12RELION3.0.2classification
13cryoSPARC2.0.53D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1063710
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 381468 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d
ELECTRON MICROSCOPYf_angle_d1.05117403
ELECTRON MICROSCOPYf_dihedral_angle_d11.0517555
ELECTRON MICROSCOPYf_chiral_restr0.0571946
ELECTRON MICROSCOPYf_plane_restr0.0081999

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