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- EMDB-25197: Cryo-EM structure of 7SK core RNP with linear RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-25197
TitleCryo-EM structure of 7SK core RNP with linear RNA
Map data7SK core RNP with linear RNA
Sample
  • Complex: 7SK linear core RNP with MePCE,Larp7 and linear 7SK RNA
    • Complex: 7SK snRNA methylphosphate capping enzyme, La-related protein 7
      • Protein or peptide: 7SK snRNA methylphosphate capping enzyme
      • Protein or peptide: La-related protein 7
    • Complex: RNA
      • RNA: Linear 7SK RNA
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE
Function / homology
Function and homology information


RNA 5'-gamma-phosphate methyltransferase activity / U6 2'-O-snRNA methylation / snRNA metabolic process / snRNA modification / 7SK snRNP / positive regulation of snRNA transcription by RNA polymerase II / 7SK snRNA binding / snRNA binding / box C/D sno(s)RNA 3'-end processing / positive regulation of protein localization to Cajal body ...RNA 5'-gamma-phosphate methyltransferase activity / U6 2'-O-snRNA methylation / snRNA metabolic process / snRNA modification / 7SK snRNP / positive regulation of snRNA transcription by RNA polymerase II / 7SK snRNA binding / snRNA binding / box C/D sno(s)RNA 3'-end processing / positive regulation of protein localization to Cajal body / RNA methyltransferase activity / RNA methylation / : / negative regulation of viral transcription / S-adenosylmethionine-dependent methyltransferase activity / O-methyltransferase activity / regulation of mRNA splicing, via spliceosome / negative regulation of transcription elongation by RNA polymerase II / positive regulation of G1/S transition of mitotic cell cycle / U6 snRNA binding / Transferases; Transferring one-carbon groups; Methyltransferases / RNA splicing / mRNA processing / spermatogenesis / cell differentiation / ribonucleoprotein complex / mRNA binding / negative regulation of transcription by RNA polymerase II / RNA binding / nucleoplasm / nucleus / cytosol
Similarity search - Function
LARP7, RNA recognition motif 1 / LARP7, RNA recognition motif 2 / La-related protein 7, La domain / RNA methyltransferase bin3, C-terminal / Bin3-type S-adenosyl-L-methionine binding domain / RNA methyltransferase Bin3-like / Bicoid-interacting protein 3 (Bin3) / Bin3-type S-adenosyl-L-methionine (SAM) domain profile. / RNA binding motif / Lupus La protein ...LARP7, RNA recognition motif 1 / LARP7, RNA recognition motif 2 / La-related protein 7, La domain / RNA methyltransferase bin3, C-terminal / Bin3-type S-adenosyl-L-methionine binding domain / RNA methyltransferase Bin3-like / Bicoid-interacting protein 3 (Bin3) / Bin3-type S-adenosyl-L-methionine (SAM) domain profile. / RNA binding motif / Lupus La protein / xRRM domain profile. / La protein, xRRM domain / La domain containing protein / La domain / Domain in the RNA-binding Lupus La protein; unknown function / La-type HTH domain / La-type HTH domain profile. / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / RNA-binding domain superfamily / Winged helix DNA-binding domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / Winged helix-like DNA-binding domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
La-related protein 7 / 7SK snRNA methylphosphate capping enzyme
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsYang Y / Liu S / Zhou ZH / Feigon J
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131901 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI155170 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM071940 United States
American Heart Association20POST35210850 United States
CitationJournal: Mol Cell / Year: 2022
Title: Structural basis of RNA conformational switching in the transcriptional regulator 7SK RNP.
Authors: Yuan Yang / Shiheng Liu / Sylvain Egloff / Catherine D Eichhorn / Tanya Hadjian / James Zhen / Tamás Kiss / Z Hong Zhou / Juli Feigon /
Abstract: 7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is ...7SK non-coding RNA (7SK) negatively regulates RNA polymerase II (RNA Pol II) elongation by inhibiting positive transcription elongation factor b (P-TEFb), and its ribonucleoprotein complex (RNP) is hijacked by HIV-1 for viral transcription and replication. Methylphosphate capping enzyme (MePCE) and La-related protein 7 (Larp7) constitutively associate with 7SK to form a core RNP, while P-TEFb and other proteins dynamically assemble to form different complexes. Here, we present the cryo-EM structures of 7SK core RNP formed with two 7SK conformations, circular and linear, and uncover a common RNA-dependent MePCE-Larp7 complex. Together with NMR, biochemical, and cellular data, these structures reveal the mechanism of MePCE catalytic inactivation in the core RNP, unexpected interactions between Larp7 and RNA that facilitate a role as an RNP chaperone, and that MePCE-7SK-Larp7 core RNP serves as a scaffold for switching between different 7SK conformations essential for RNP assembly and regulation of P-TEFb sequestration and release.
History
DepositionOct 24, 2021-
Header (metadata) releaseMar 30, 2022-
Map releaseMar 30, 2022-
UpdateMay 18, 2022-
Current statusMay 18, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25197.png

Downloads & links

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Map

FileDownload / File: emd_25197.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation7SK core RNP with linear RNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 200 pix.
= 214. Å		1.07 Å/pix.
x 200 pix.
= 214. Å		1.07 Å/pix.
x 200 pix.
= 214. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.13048209 - 0.15125369
Average (Standard dev.)-1.340885e-06 (±0.005138106)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 214.00002 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: 7SK core RNP with linear RNA

Fileemd_25197_additional_1.map
Annotation7SK core RNP with linear RNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 7SK linear core RNP with MePCE,Larp7 and linear 7SK RNA

EntireName: 7SK linear core RNP with MePCE,Larp7 and linear 7SK RNA
Components
  • Complex: 7SK linear core RNP with MePCE,Larp7 and linear 7SK RNA
    • Complex: 7SK snRNA methylphosphate capping enzyme, La-related protein 7
      • Protein or peptide: 7SK snRNA methylphosphate capping enzyme
      • Protein or peptide: La-related protein 7
    • Complex: RNA
      • RNA: Linear 7SK RNA
  • Ligand: S-ADENOSYL-L-HOMOCYSTEINE

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Supramolecule #1: 7SK linear core RNP with MePCE,Larp7 and linear 7SK RNA

SupramoleculeName: 7SK linear core RNP with MePCE,Larp7 and linear 7SK RNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: 7SK snRNA methylphosphate capping enzyme, La-related protein 7

SupramoleculeName: 7SK snRNA methylphosphate capping enzyme, La-related protein 7
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21(DE3)

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3

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Macromolecule #1: 7SK snRNA methylphosphate capping enzyme

MacromoleculeName: 7SK snRNA methylphosphate capping enzyme / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Transferring one-carbon groups; Methyltransferases
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 35.122797 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSP LPAAGFKKQQ RKFQYGNYCK YYGYRNPSCE DGRLRVLKPE WFRGRDVLDL GCNVGHLTLS IACKWGPSR MVGLDIDSRL IHSARQNIRH YLSEELRLPP QTLEGDPGAE GEEGTTTVRK RSCFPASLTA SRGPIAAPQV P LDGADTSV ...String:
MGSSHHHHHH SSGLVPRGSP LPAAGFKKQQ RKFQYGNYCK YYGYRNPSCE DGRLRVLKPE WFRGRDVLDL GCNVGHLTLS IACKWGPSR MVGLDIDSRL IHSARQNIRH YLSEELRLPP QTLEGDPGAE GEEGTTTVRK RSCFPASLTA SRGPIAAPQV P LDGADTSV FPNNVVFVTG NYVLDRDDLV EAQTPEYDVV LCLSLTKWVH LNWGDEGLKR MFRRIYRHLR PGGILVLEPQ PW SSYGKRK TLTETIYKNY YRIQLKPEQF SSYLTSPDVG FSSYELVATP HNTSKGFQRP VYLFHKARSP SH

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Macromolecule #2: La-related protein 7

MacromoleculeName: La-related protein 7 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 55.109129 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GMETESGNQE KVMEEESTEK KKEVEKKKRS RVKQVLADIA KQVDFWFGDA NLHKDRFLRE QIEKSRDGYV DISLLVSFNK MKKLTTDGK LIARALRSSA VVELDLEGTR IRRKKPLGER PKDEDERTVY VELLPKNVNH SWIERVFGKC GNVVYISIPH Y KSTGDPKG ...String:
GMETESGNQE KVMEEESTEK KKEVEKKKRS RVKQVLADIA KQVDFWFGDA NLHKDRFLRE QIEKSRDGYV DISLLVSFNK MKKLTTDGK LIARALRSSA VVELDLEGTR IRRKKPLGER PKDEDERTVY VELLPKNVNH SWIERVFGKC GNVVYISIPH Y KSTGDPKG FAFVEFETKE QAAKAIEFLN NPPEEAPRKP GIFPKTVKNK PIPALRVVEE KKKKKKKKGR MKKEDNIQAK EE NMDTSNT SISKMKRSRP TSEGSDIEST GEEVIPLRVL SKSEWMDLKK EYLALQKASM ASLKKTISQI KSESEMETDS GVP QNTGMK NEKTANREEC RTQEKVNATG PQFVSGVIVK IISTEPLPGR KQVRDTLAAI SEVLYVDLLE GDTECHARFK TPED AQAVI NAYTEINKKH CWKLEILSGD HEQRYWQKIL VDRQAKLNQP REKKRGTEKL ITKAEKIRLA KTQQASKHIR FSEYD

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Macromolecule #3: Linear 7SK RNA

MacromoleculeName: Linear 7SK RNA / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 26.09623 KDa
SequenceString:
(G5J)GAUGUGAGG GCGACUUCGG UCCUCCCUCA CCGCUCCAUG UGCGAAAUGA GGCGCUGCAU GUGGCAGUCU GCCUUU CUU UU

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Macromolecule #4: S-ADENOSYL-L-HOMOCYSTEINE

MacromoleculeName: S-ADENOSYL-L-HOMOCYSTEINE / type: ligand / ID: 4 / Number of copies: 1 / Formula: SAH
Molecular weightTheoretical: 384.411 Da
Chemical component information

ChemComp-SAH:
S-ADENOSYL-L-HOMOCYSTEINE / S-Adenosyl-L-homocysteine

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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 / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 159349

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