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- PDB-7z52: Human NEXT dimer - focused reconstruction of the single MTR4 -

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Basic information

Entry
Database: PDB / ID: 7z52
TitleHuman NEXT dimer - focused reconstruction of the single MTR4
Components
  • Exosome RNA helicase MTR4
  • RNA (5'-R(P*UP*UP*UP*UP*U)-3')
  • Zinc finger CCHC domain-containing protein 8
KeywordsRNA BINDING PROTEIN / HELICASE / ATPASE / RNA DEGRADATION / EXOSOME
Function / homology
Function and homology information


snRNA catabolic process / TRAMP complex / mRNA 3'-end processing / RNA catabolic process / maturation of 5.8S rRNA / Major pathway of rRNA processing in the nucleolus and cytosol / RNA processing / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex ...snRNA catabolic process / TRAMP complex / mRNA 3'-end processing / RNA catabolic process / maturation of 5.8S rRNA / Major pathway of rRNA processing in the nucleolus and cytosol / RNA processing / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / Regulation of endogenous retroelements by the Human Silencing Hub (HUSH) complex / mRNA splicing, via spliceosome / rRNA processing / RNA helicase activity / nuclear body / RNA helicase / nuclear speck / DNA damage response / nucleolus / ATP hydrolysis activity / RNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus
Similarity search - Function
: / rRNA-processing arch domain / Mtr4-like, beta-barrel domain / ATP-dependent RNA helicase Ski2, C-terminal / ATP-dependent RNA helicase Ski2-like / : / DSHCT (NUC185) domain / Exosome RNA helicase MTR4-like, stalk / DSHCT / : ...: / rRNA-processing arch domain / Mtr4-like, beta-barrel domain / ATP-dependent RNA helicase Ski2, C-terminal / ATP-dependent RNA helicase Ski2-like / : / DSHCT (NUC185) domain / Exosome RNA helicase MTR4-like, stalk / DSHCT / : / PSP, proline-rich / PSP / proline-rich domain in spliceosome associated proteins / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / zinc finger / Zinc knuckle / Zinc finger, CCHC-type / Zinc finger CCHC-type profile. / 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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / RNA / Exosome RNA helicase MTR4 / Zinc finger CCHC domain-containing protein 8
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsGerlach, P. / Lingaraju, M. / Salerno-Kochan, A. / Bonneau, F. / Basquin, J. / Conti, E.
Funding support Germany, European Union, 4items
OrganizationGrant numberCountry
Max Planck Society Germany
European CommissionERC-2016-ADG 740329 EXORICOEuropean Union
German Research Foundation (DFG)SFB/TRR 237 Germany
German Research Foundation (DFG)SFB 1035 Germany
CitationJournal: Mol Cell / Year: 2022
Title: Structure and regulation of the nuclear exosome targeting complex guides RNA substrates to the exosome.
Authors: Piotr Gerlach / William Garland / Mahesh Lingaraju / Anna Salerno-Kochan / Fabien Bonneau / Jérôme Basquin / Torben Heick Jensen / Elena Conti /
Abstract: In mammalian cells, spurious transcription results in a vast repertoire of unproductive non-coding RNAs, whose deleterious accumulation is prevented by rapid decay. The nuclear exosome targeting ...In mammalian cells, spurious transcription results in a vast repertoire of unproductive non-coding RNAs, whose deleterious accumulation is prevented by rapid decay. The nuclear exosome targeting (NEXT) complex plays a central role in directing non-functional transcripts to exosome-mediated degradation, but the structural and molecular mechanisms remain enigmatic. Here, we elucidated the architecture of the human NEXT complex, showing that it exists as a dimer of MTR4-ZCCHC8-RBM7 heterotrimers. Dimerization preconfigures the major MTR4-binding region of ZCCHC8 and arranges the two MTR4 helicases opposite to each other, with each protomer able to function on many types of RNAs. In the inactive state of the complex, the 3' end of an RNA substrate is enclosed in the MTR4 helicase channel by a ZCCHC8 C-terminal gatekeeping domain. The architecture of a NEXT-exosome assembly points to the molecular and regulatory mechanisms with which the NEXT complex guides RNA substrates to the exosome.
History
DepositionMar 7, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 22, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 20, 2022Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Jul 17, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Exosome RNA helicase MTR4
R: RNA (5'-R(P*UP*UP*UP*UP*U)-3')
A: Zinc finger CCHC domain-containing protein 8
hetero molecules


Theoretical massNumber of molelcules
Total (without water)199,1364
Polymers198,6293
Non-polymers5061
Water00
1


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

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Components

#1: Protein Exosome RNA helicase MTR4 / ATP-dependent RNA helicase DOB1 / ATP-dependent RNA helicase SKIV2L2 / Superkiller viralicidic ...ATP-dependent RNA helicase DOB1 / ATP-dependent RNA helicase SKIV2L2 / Superkiller viralicidic activity 2-like 2 / TRAMP-like complex helicase


Mass: 118322.070 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MTREX, DOB1, KIAA0052, MTR4, SKIV2L2 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42285, RNA helicase
#2: RNA chain RNA (5'-R(P*UP*UP*UP*UP*U)-3')


Mass: 1485.872 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Protein Zinc finger CCHC domain-containing protein 8 / TRAMP-like complex RNA-binding factor ZCCHC8


Mass: 78821.375 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ZCCHC8 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q6NZY4
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
Has ligand of interestY

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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 NEXT dimer - focused reconstruction of the single MTR4
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE

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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 defocus max: 2300 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 1.705 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 224200 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0046030
ELECTRON MICROSCOPYf_angle_d0.5978177
ELECTRON MICROSCOPYf_dihedral_angle_d10.179865
ELECTRON MICROSCOPYf_chiral_restr0.042940
ELECTRON MICROSCOPYf_plane_restr0.0041033

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