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- PDB-6lxe: DROSHA-DGCR8 complex -

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Basic information

Entry
Database: PDB / ID: 6lxe
TitleDROSHA-DGCR8 complex
Components
  • Microprocessor complex subunit DGCR8
  • Ribonuclease 3
KeywordsHYDROLASE/RNA BINDING PROTEIN / Ribonuclease / RNA BINDING PROTEIN / HYDROLASE-RNA BINDING PROTEIN 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsJin, W. / Wang, J. / Liu, C.P. / Wang, H.W. / Xu, R.M.
Funding support China, 6items
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, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 15, 2020Provider: repository / Type: Initial release
Revision 1.1May 27, 2020Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Mar 27, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Ribonuclease 3
B: Microprocessor complex subunit DGCR8
C: Microprocessor complex subunit DGCR8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)287,8645
Polymers287,7333
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2540 Å2
ΔGint-20 kcal/mol
Surface area43480 Å2

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Components

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


Mass: 115390.578 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9NRR4, ribonuclease III
#2: Protein Microprocessor complex subunit DGCR8 / / DiGeorge syndrome critical region 8


Mass: 86171.203 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: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q8WYQ5
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN

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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: Ternary complex of DROSHA-DGCR8 / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 109333 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 5B16

5b16


Accession code: 5B16 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026828
ELECTRON MICROSCOPYf_angle_d0.5379228
ELECTRON MICROSCOPYf_dihedral_angle_d8.6864145
ELECTRON MICROSCOPYf_chiral_restr0.0381001
ELECTRON MICROSCOPYf_plane_restr0.0031197

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