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- EMDB-14513: Human NEXT dimer - focused reconstruction of the single MTR4 -

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Basic information

Entry
Database: EMDB / ID: EMD-14513
TitleHuman NEXT dimer - focused reconstruction of the single MTR4
Map dataNEXT_L_focused_on_MTR4_postprocess
Sample
  • Complex: Human NEXT dimer - focused reconstruction of the single MTR4
    • Protein or peptide: Exosome RNA helicase MTR4
    • RNA: RNA (5'-R(P*UP*UP*UP*UP*U)-3')
    • Protein or peptide: Zinc finger CCHC domain-containing protein 8
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Function / homology
Function and homology information


: / snRNA catabolic process / TRAMP complex / RNA catabolic process / maturation of 5.8S rRNA / Major pathway of rRNA processing in the nucleolus and cytosol / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / mRNA splicing, via spliceosome / rRNA processing ...: / snRNA catabolic process / TRAMP complex / RNA catabolic process / maturation of 5.8S rRNA / Major pathway of rRNA processing in the nucleolus and cytosol / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / 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 / : / Exosome RNA helicase MTR4-like, stalk / ATP-dependent RNA helicase Ski2, C-terminal / ATP-dependent RNA helicase Ski2-like / DSHCT (NUC185) domain / DSHCT / PSP, proline-rich / PSP ...rRNA-processing arch domain / Mtr4-like, beta-barrel domain / : / Exosome RNA helicase MTR4-like, stalk / ATP-dependent RNA helicase Ski2, C-terminal / ATP-dependent RNA helicase Ski2-like / DSHCT (NUC185) domain / DSHCT / PSP, proline-rich / PSP / proline-rich domain in spliceosome associated proteins / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / 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
Exosome RNA helicase MTR4 / Zinc finger CCHC domain-containing protein 8
Similarity search - Component
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle 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, 4 items
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, 2022-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateJul 20, 2022-
Current statusJul 20, 2022Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14513.png

Downloads & links

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Map

FileDownload / File: emd_14513.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNEXT_L_focused_on_MTR4_postprocess
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 256 pix.
= 217.907 Å		0.85 Å/pix.
x 256 pix.
= 217.907 Å		0.85 Å/pix.
x 256 pix.
= 217.907 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8512 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.08104938 - 0.13842277
Average (Standard dev.)0.00016477695 (±0.0032759518)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.9072 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14513_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: NEXT L focused on MTR4 refine 741 denmod map

Fileemd_14513_additional_1.map
AnnotationNEXT_L_focused_on_MTR4_refine_741_denmod_map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: NEXT L focused on MTR4 half map 1

Fileemd_14513_half_map_1.map
AnnotationNEXT_L_focused_on_MTR4_half_map_1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: NEXT L focused on MTR4 half map 2

Fileemd_14513_half_map_2.map
AnnotationNEXT_L_focused_on_MTR4_half_map_2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human NEXT dimer - focused reconstruction of the single MTR4

EntireName: Human NEXT dimer - focused reconstruction of the single MTR4
Components
  • Complex: Human NEXT dimer - focused reconstruction of the single MTR4
    • Protein or peptide: Exosome RNA helicase MTR4
    • RNA: RNA (5'-R(P*UP*UP*UP*UP*U)-3')
    • Protein or peptide: Zinc finger CCHC domain-containing protein 8
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: Human NEXT dimer - focused reconstruction of the single MTR4

SupramoleculeName: Human NEXT dimer - focused reconstruction of the single MTR4
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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Macromolecule #1: Exosome RNA helicase MTR4

MacromoleculeName: Exosome RNA helicase MTR4 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 118.32207 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GPDSMADAFG DELFSVFEGD STTAAGTKKD KEKDKGKWKG PPGSADKAGK RFDGKLQSES TNNGKNKRDV DFEGTDEPIF GKKPRIEES ITEDLSLADL MPRVKVQSVE TVEGCTHEVA LPAEEDYLPL KPRVGKAAKE YPFILDAFQR EAIQCVDNNQ S VLVSAHTS ...String:
GPDSMADAFG DELFSVFEGD STTAAGTKKD KEKDKGKWKG PPGSADKAGK RFDGKLQSES TNNGKNKRDV DFEGTDEPIF GKKPRIEES ITEDLSLADL MPRVKVQSVE TVEGCTHEVA LPAEEDYLPL KPRVGKAAKE YPFILDAFQR EAIQCVDNNQ S VLVSAHTS AGKTVCAEYA IALALREKQR VIFTSPIKAL SNQKYREMYE EFQDVGLMTG DVTINPTASC LVMTTEILRS ML YRGSEVM REVAWVIFDE IHYMRDSERG VVWEETIILL PDNVHYVFLS ATIPNARQFA EWICHLHKQP CHVIYTDYRP TPL QHYIFP AGGDGLHLVV DENGDFREDN FNTAMQVLRD AGDLAKGDQK GRKGGTKGPS NVFKIVKMIM ERNFQPVIIF SFSK KDCEA YALQMTKLDF NTDEEKKMVE EVFSNAIDCL SDEDKKLPQV EHVLPLLKRG IGIHHGGLLP ILKETIEILF SEGLI KALF ATETFAMGIN MPARTVLFTN ARKFDGKDFR WISSGEYIQM SGRAGRRGMD DRGIVILMVD EKMSPTIGKQ LLKGSA DPL NSAFHLTYNM VLNLLRVEEI NPEYMLEKSF YQFQHYRAIP GVVEKVKNSE EQYNKIVIPN EESVVIYYKI RQQLAKL GK EIEEYIHKPK YCLPFLQPGR LVKVKNEGDD FGWGVVVNFS KKSNVKPNSG ELDPLYVVEV LLRCSKESLK NSATEAAK P AKPDEKGEMQ VVPVLVHLLS AISSVRLYIP KDLRPVDNRQ SVLKSIQEVQ KRFPDGIPLL DPIDDMGIQD QGLKKVIQK VEAFEHRMYS HPLHNDPNLE TVYTLCEKKA QIAIDIKSAK RELKKARTVL QMDELKCRKR VLRRLGFATS SDVIEMKGRV ACEISSADE LLLTEMMFNG LFNDLSAEQA TALLSCFVFQ ENSSEMPKLT EQLAGPLRQM QECAKRIAKV SAEAKLEIDE E TYLSSFKP HLMDVVYTWA TGATFAHICK MTDVFEGSII RCMRRLEELL RQMCQAAKAI GNTELENKFA EGITKIKRDI VF AASLYL

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Macromolecule #3: Zinc finger CCHC domain-containing protein 8

MacromoleculeName: Zinc finger CCHC domain-containing protein 8 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 78.821375 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: RSMAAEVYFG DLELFEPFDH PGESIPKPVH TRFKDDDGDE EDENGVGDAE LRERLRQCEE TIEQLRAENQ ELKRKLNILT RPSGILVND TKLDGPILQI LFMNNAISKQ YHQEIEEFVS NLVKRFEEQQ KNDVEKTSFN LLPQPSSIVL EEDHKVEESC A IKNNKEAF ...String:
RSMAAEVYFG DLELFEPFDH PGESIPKPVH TRFKDDDGDE EDENGVGDAE LRERLRQCEE TIEQLRAENQ ELKRKLNILT RPSGILVND TKLDGPILQI LFMNNAISKQ YHQEIEEFVS NLVKRFEEQQ KNDVEKTSFN LLPQPSSIVL EEDHKVEESC A IKNNKEAF SVVGSVLYFT NFCLDKLGQP LLNENPQLSE GWEIPKYHQV FSHIVSLEGQ EIQVKAKRPK PHCFNCGSEE HQ MKDCPMP RNAARISEKR KEYMDACGEA NNQNFQQRYH AEEVEERFGR FKPGVISEEL QDALGVTDKS LPPFIYRMRQ LGY PPGWLK EAELENSGLA LYDGKDGTDG ETEVGEIQQN KSVTYDLSKL VNYPGFNIST PRGIPDEWRI FGSIPMQACQ QKDV FANYL TSNFQAPGVK SGNKRSSSHS SPGSPKKQKN ESNSAGSPAD MELDSDMEVP HGSQSSESFQ FQPPLPPDTP PLPRG TPPP VFTPPLPKGT PPLTPSDSPQ TRTGSGAVDE DALTLEELEE QQRRIWAALE QAESVNSDSD VPVDTPLTGN SVASSP CPN ELDLPVPEGK TSEKQTLDEP EVPEIFTKKS EAGHASSPDS EVTSLCQKEK AELAPVNTEG ALLDNGSVVP NCDISNG GS QKLFPADTSP STATKIHSPI PDMSKFATGI TPFEFENMAE STGMYLRIRS LLKNSPRNQQ KNKKASE

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Macromolecule #2: RNA (5'-R(P*UP*UP*UP*UP*U)-3')

MacromoleculeName: RNA (5'-R(P*UP*UP*UP*UP*U)-3') / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.485872 KDa
SequenceString:
UUUUU

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 1 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.705 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 224200
FSC plot (resolution estimation)

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