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- PDB-5zal: Cryo-EM structure of human Dicer and its complexes with a pre-miR... -

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Basic information

Entry
Database: PDB / ID: 5zal
TitleCryo-EM structure of human Dicer and its complexes with a pre-miRNA substrate
Components
  • Endoribonuclease Dicer
  • RISC-loading complex subunit TARBP2
  • RNA (73-mer)
KeywordsHYDROLASE/PROTEIN BINDING/RNA / Dicer / TRBP / Cryo-EM / RNA interference / PROTEIN BINDING / HYDROLASE-PROTEIN BINDING-RNA complex
Function / homology
Function and homology information


regulation of siRNA processing / regulation of miRNA processing / negative regulation of cytoplasmic pattern recognition receptor signaling pathway / regulation of viral transcription / peripheral nervous system myelin formation / regulation of regulatory ncRNA processing / negative regulation of defense response to virus by host / global gene silencing by mRNA cleavage / pre-miRNA binding / negative regulation of Schwann cell proliferation ...regulation of siRNA processing / regulation of miRNA processing / negative regulation of cytoplasmic pattern recognition receptor signaling pathway / regulation of viral transcription / peripheral nervous system myelin formation / regulation of regulatory ncRNA processing / negative regulation of defense response to virus by host / global gene silencing by mRNA cleavage / pre-miRNA binding / negative regulation of Schwann cell proliferation / tRNA-derived small RNA (tsRNA or tRNA-related fragment, tRF) biogenesis / Small interfering RNA (siRNA) biogenesis / apoptotic DNA fragmentation / tRNA decay / ribonuclease III / deoxyribonuclease I activity / positive regulation of Schwann cell differentiation / nerve development / RISC-loading complex / positive regulation of myelination / miRNA metabolic process / RISC complex assembly / miRNA processing / ribonuclease III activity / skeletal muscle tissue regeneration / pre-miRNA processing / siRNA processing / siRNA binding / Regulation of MITF-M-dependent genes involved in apoptosis / M-decay: degradation of maternal mRNAs by maternally stored factors / pre-mRNA binding / RISC complex / miRNA binding / neural precursor cell proliferation / MicroRNA (miRNA) biogenesis / positive regulation of muscle cell differentiation / negative regulation of tumor necrosis factor production / spermatid development / negative regulation of tumor necrosis factor-mediated signaling pathway / single fertilization / positive regulation of viral genome replication / negative regulation of protein kinase activity / neuron projection morphogenesis / RNA endonuclease activity / protein sequestering activity / positive regulation of translation / helicase activity / PKR-mediated signaling / multicellular organism growth / double-stranded RNA binding / nuclear body / protein domain specific binding / negative regulation of gene expression / perinuclear region of cytoplasm / enzyme binding / negative regulation of transcription by RNA polymerase II / protein homodimerization activity / RNA binding / extracellular exosome / nucleoplasm / ATP binding / metal ion binding / identical protein binding / nucleus / cytoplasm / cytosol
Similarity search - Function
RISC-loading complex subunit TRBP2 / TRBP2 , first double-stranded RNA binding domain / TRBP2 , second double-stranded RNA binding domain / TRBP2 , third double-stranded RNA binding domain / Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / : / : ...RISC-loading complex subunit TRBP2 / TRBP2 , first double-stranded RNA binding domain / TRBP2 , second double-stranded RNA binding domain / TRBP2 , third double-stranded RNA binding domain / Dicer, double-stranded RNA-binding domain / : / Dicer, partner-binding domain / Dicer, dsRNA-binding domain / : / : / Dicer, platform domain / Dicer dimerisation domain / Dicer dimerisation domain superfamily / Dicer dimerisation domain / Dicer double-stranded RNA-binding fold domain profile. / Ribonuclease III family signature. / Ribonuclease III domain / Ribonuclease III family domain profile. / Ribonuclease III family / Ribonuclease III domain / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain profile. / PAZ domain / Type III restriction enzyme, res subunit / 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 / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / RISC-loading complex subunit TARBP2 / Endoribonuclease Dicer
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.7 Å
AuthorsLiu, Z. / Wang, J. / Cheng, H. / Ke, X. / Sun, L. / Zhang, Q.C. / Wang, H.-W.
Funding support China, 1items
OrganizationGrant numberCountry
National Science Foundation of China31530018 China
CitationJournal: Cell / Year: 2018
Title: Cryo-EM Structure of Human Dicer and Its Complexes with a Pre-miRNA Substrate.
Authors: Zhongmin Liu / Jia Wang / Hang Cheng / Xin Ke / Lei Sun / Qiangfeng Cliff Zhang / Hong-Wei Wang /
Abstract: Human Dicer (hDicer) is a multi-domain protein belonging to the RNase III family. It plays pivotal roles in small RNA biogenesis during the RNA interference (RNAi) pathway by processing a diverse ...Human Dicer (hDicer) is a multi-domain protein belonging to the RNase III family. It plays pivotal roles in small RNA biogenesis during the RNA interference (RNAi) pathway by processing a diverse range of double-stranded RNA (dsRNA) precursors to generate ∼22 nt microRNA (miRNA) or small interfering RNA (siRNA) products for sequence-directed gene silencing. In this work, we solved the cryoelectron microscopy (cryo-EM) structure of hDicer in complex with its cofactor protein TRBP and revealed the precise spatial arrangement of hDicer's multiple domains. We further solved structures of the hDicer-TRBP complex bound with pre-let-7 RNA in two distinct conformations. In combination with biochemical analysis, these structures reveal a property of the hDicer-TRBP complex to promote the stability of pre-miRNA's stem duplex in a pre-dicing state. These results provide insights into the mechanism of RNA processing by hDicer and illustrate the regulatory role of hDicer's N-terminal helicase domain.
History
DepositionFeb 7, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0May 9, 2018Provider: repository / Type: Initial release
Revision 1.1Aug 8, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_id_ISSN / _citation.journal_volume ..._citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Nov 6, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB
Revision 1.3Aug 5, 2020Group: Data collection / Category: em_imaging_optics / Item: _em_imaging_optics.phase_plate
Revision 1.4Oct 16, 2024Group: Data collection / Database references ...Data collection / Database references / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature
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 / _em_admin.last_update

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Structure visualization

Movie
  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-6905
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Endoribonuclease Dicer
B: RISC-loading complex subunit TARBP2
C: RNA (73-mer)


Theoretical massNumber of molelcules
Total (without water)281,4083
Polymers281,4083
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4940 Å2
ΔGint-39 kcal/mol
Surface area76070 Å2

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Components

#1: Protein Endoribonuclease Dicer / Helicase with RNase motif / Helicase MOI


Mass: 218947.328 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: DICER1, DICER, HERNA, KIAA0928 / Production host: Homo sapiens (human) / References: UniProt: Q9UPY3, ribonuclease III
#2: Protein RISC-loading complex subunit TARBP2 / TTRBP / AR RNA-binding protein 2 / Trans-activation-responsive RNA-binding protein


Mass: 39085.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TARBP2, TRBP / Production host: Homo sapiens (human) / References: UniProt: Q15633
#3: RNA chain RNA (73-mer) / Prelet7


Mass: 23375.830 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: cell-free synthesis (others)
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Dicer and trbpCOMPLEXall0RECOMBINANT
2Human Dicer-TRBP with Pre-let-7 complex Class ICOMPLEXall1RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11
210.27 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Homo sapiens (human)9606
32Homo sapiens (human)9606
Buffer solutionpH: 8
Details: 50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM DTT, 2 mM CaCl2
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse.
Specimen supportDetails: The grid was glow-discharged prior to use. / Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: no
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 89000 X / Calibrated magnification: 36496 X / Nominal defocus max: 3500 nm / Nominal defocus min: 2500 nm / Calibrated defocus min: 2500 nm / Calibrated defocus max: 3500 nm / Cs: 0.01 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 5.44 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2849 / Details: no
EM imaging opticsPhase plate: OTHER
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 32 / Used frames/image: 1-32

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
3RELION2.1CTF correction
6UCSF Chimera1.1model fitting
8PHENIX1.11model refinement
9RELION2.1initial Euler assignment
10RELION2.1final Euler assignment
11RELION2.1classification
12RELION2.13D reconstruction
Image processingDetails: no
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 130000 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 100 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: CC
Atomic model buildingPDB-ID: 5ZAK

5zak


Pdb chain-ID: A / Accession code: 5ZAK / Pdb chain residue range: 1-1922 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00812712
ELECTRON MICROSCOPYf_angle_d1.41117470
ELECTRON MICROSCOPYf_dihedral_angle_d9.667662
ELECTRON MICROSCOPYf_chiral_restr0.0682036
ELECTRON MICROSCOPYf_plane_restr0.012012

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