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- EMDB-6581: 3D structure determination of the human *U4/U6.U5* tri-snRNP complex -

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Entry
Database: EMDB / ID: EMD-6581
Title3D structure determination of the human *U4/U6.U5* tri-snRNP complex
Map dataReconstruction of human tri-snRNP
Sample
  • Sample: human U4/U6.U5 tri-snRNP
  • Protein or peptide: U4/U6.U5 tri-snRNP
KeywordssnRNP / splicing / spliceosome / human
Function / homology
Function and homology information


Lsm2-8 complex / U6 snRNA 3'-end binding / spliceosomal snRNP complex / ribonucleoprotein complex localization / snRNP binding / U4atac snRNP / RNA localization / R-loop processing / U4atac snRNA binding / mRNA decay by 5' to 3' exoribonuclease ...Lsm2-8 complex / U6 snRNA 3'-end binding / spliceosomal snRNP complex / ribonucleoprotein complex localization / snRNP binding / U4atac snRNP / RNA localization / R-loop processing / U4atac snRNA binding / mRNA decay by 5' to 3' exoribonuclease / Lsm1-7-Pat1 complex / U6 snRNP / box C/D sno(s)RNA binding / positive regulation of primary miRNA processing / PH domain binding / U2 snRNP binding / U7 snRNA binding / histone pre-mRNA DCP binding / U7 snRNP / dense fibrillar component / histone pre-mRNA 3'end processing complex / cis assembly of pre-catalytic spliceosome / SLBP independent Processing of Histone Pre-mRNAs / SLBP Dependent Processing of Replication-Dependent Histone Pre-mRNAs / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / protein methylation / U12-type spliceosomal complex / methylosome / P-body assembly / U4/U6 snRNP / 7-methylguanosine cap hypermethylation / U1 snRNP binding / pICln-Sm protein complex / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / small nuclear ribonucleoprotein complex / sno(s)RNA-containing ribonucleoprotein complex / SMN-Sm protein complex / spliceosomal tri-snRNP complex / P granule / U4 snRNA binding / telomerase holoenzyme complex / box C/D methylation guide snoRNP complex / U2-type spliceosomal complex / U2-type precatalytic spliceosome / commitment complex / telomerase RNA binding / U2-type prespliceosome assembly / U2-type catalytic step 2 spliceosome / U4 snRNP / box C/D snoRNP assembly / U2 snRNP / rRNA modification in the nucleus and cytosol / RNA Polymerase II Transcription Termination / tRNA processing / U1 snRNP / U3 snoRNA binding / nuclear-transcribed mRNA catabolic process / U2-type prespliceosome / K63-linked polyubiquitin modification-dependent protein binding / positive regulation of miRNA metabolic process / precatalytic spliceosome / spliceosomal complex assembly / mRNA Splicing - Minor Pathway / mRNA catabolic process / MLL1 complex / spliceosomal tri-snRNP complex assembly / single fertilization / Major pathway of rRNA processing in the nucleolus and cytosol / U5 snRNA binding / U5 snRNP / RNA processing / U2 snRNA binding / U6 snRNA binding / spliceosomal snRNP assembly / Cajal body / ribonucleoprotein complex binding / pre-mRNA intronic binding / U1 snRNA binding / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / helicase activity / response to cocaine / maturation of LSU-rRNA / maturation of SSU-rRNA / small-subunit processome / response to bacterium / spliceosomal complex / P-body / mRNA processing / small GTPase binding / mRNA splicing, via spliceosome / osteoblast differentiation / cellular response to xenobiotic stimulus / protein-macromolecule adaptor activity / cellular response to tumor necrosis factor / ribosomal small subunit biogenesis / snRNP Assembly
Similarity search - Function
USP39 / PRP4-like superfamily / pre-mRNA processing factor 4 (PRP4) like / PWI domain superfamily / PWI domain / PWI domain profile. / Sm-like protein Lsm8 / U6 snRNA-associated Sm-like protein Lsm1/8 / Pre-mRNA processing factor 4 (PRP4)-like / U6 snRNA-associated Sm-like protein LSm2 ...USP39 / PRP4-like superfamily / pre-mRNA processing factor 4 (PRP4) like / PWI domain superfamily / PWI domain / PWI domain profile. / Sm-like protein Lsm8 / U6 snRNA-associated Sm-like protein Lsm1/8 / Pre-mRNA processing factor 4 (PRP4)-like / U6 snRNA-associated Sm-like protein LSm2 / Sm-like protein Lsm4 / Splicing Factor Motif, present in Prp18 and Pr04 / Sm-like protein Lsm7 / Sm-like protein LSm5 / Sm-like protein Lsm3 / U6 snRNA-associated Sm-like protein Lsm3 / Pre-mRNA-splicing factor 3 / U4/U6 small nuclear ribonucleoprotein Prp3 / pre-mRNA processing factor 3 domain / Prp31 C-terminal / U4/U6 small nuclear ribonucleoprotein Prp31 / Prp31 C terminal domain / Small nuclear ribonucleoprotein Prp3, C-terminal domain / Small nuclear ribonucleoprotein Prp3, C-terminal domain / PWI domain / PWI, domain in splicing factors / Dim1 family / Mitosis protein DIM1 / Mitosis protein DIM1 / PRP1 splicing factor, N-terminal / PRP1 splicing factor, N-terminal / : / Small ribonucleoprotein associated, SmB/SmN / Small nuclear ribonucleoprotein D1 / Brr2, N-terminal helicase PWI domain / : / N-terminal helicase PWI domain / Pre-mRNA-splicing helicase BRR2 plug domain / H/ACA ribonucleoprotein complex, subunit Nhp2-like / Ubiquitin Carboxyl-terminal Hydrolase-like zinc finger / Zinc finger, UBP-type / NOSIC / Pre-mRNA-splicing factor Syf1-like / Zn-finger in ubiquitin-hydrolases and other protein / Zinc finger UBP-type profile. / NOSIC (NUC001) domain / Nop domain / Nop domain superfamily / Nop, C-terminal domain / snoRNA binding domain, fibrillarin / Nop domain profile. / Sec63 Brl domain / Snu114, GTP-binding domain / 116kDa U5 small nuclear ribonucleoprotein component, N-terminal / 116kDa U5 small nuclear ribonucleoprotein component, C-terminal / 116 kDa U5 small nuclear ribonucleoprotein component N-terminus / Tetratricopeptide repeat / Small nuclear ribonucleoprotein Sm D3 / Small nuclear ribonucleoprotein Sm D2 / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein G / Small nuclear ribonucleoprotein F / Sec63 domain / Sec63 Brl domain / Sm-like protein Lsm7/SmG / Like-Sm (LSM) domain containing protein, LSm4/SmD1/SmD3 / Sm-like protein Lsm6/SmF / LSM domain / LSM domain, eukaryotic/archaea-type / snRNP Sm proteins / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / Tetratricopeptide repeat / Peptidase C19, ubiquitin carboxyl-terminal hydrolase / Ubiquitin carboxyl-terminal hydrolase / : / Ubiquitin specific protease domain / Ubiquitin specific protease (USP) domain profile. / Sm domain profile. / DEAD-box subfamily ATP-dependent helicases signature. / ATP-dependent RNA helicase DEAD-box, conserved site / RNA helicase, DEAD-box type, Q motif / Elongation Factor G, domain II / Elongation Factor G, domain III / DEAD-box RNA helicase Q motif profile. / LSM domain superfamily / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / Translation elongation factor EFG/EF2, domain IV / Elongation factor G, domain IV / Elongation factor G, domain IV / Elongation factor G C-terminus / Elongation factor EFG, domain V-like / Elongation factor G C-terminus / EF-G domain III/V-like / PROCT domain / Prp8 RNase domain IV, fingers region / PROCT (NUC072) domain / PRO8NT domain / PROCN domain / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding
Similarity search - Domain/homology
U4/U6 small nuclear ribonucleoprotein Prp4 / U4/U6 small nuclear ribonucleoprotein Prp3 / U5 small nuclear ribonucleoprotein 200 kDa helicase / Pre-mRNA-processing factor 6 / U6 snRNA-associated Sm-like protein LSm8 / Small nuclear ribonucleoprotein-associated proteins B and B' / NHP2-like protein 1 / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein F / Small nuclear ribonucleoprotein G ...U4/U6 small nuclear ribonucleoprotein Prp4 / U4/U6 small nuclear ribonucleoprotein Prp3 / U5 small nuclear ribonucleoprotein 200 kDa helicase / Pre-mRNA-processing factor 6 / U6 snRNA-associated Sm-like protein LSm8 / Small nuclear ribonucleoprotein-associated proteins B and B' / NHP2-like protein 1 / Small nuclear ribonucleoprotein E / Small nuclear ribonucleoprotein F / Small nuclear ribonucleoprotein G / U6 snRNA-associated Sm-like protein LSm3 / U6 snRNA-associated Sm-like protein LSm6 / Small nuclear ribonucleoprotein Sm D1 / Small nuclear ribonucleoprotein Sm D2 / Small nuclear ribonucleoprotein Sm D3 / Thioredoxin-like protein 4A / 116 kDa U5 small nuclear ribonucleoprotein component / Ubiquitin carboxyl-terminal hydrolase 39 / 60 kDa U4/U6 snRNP-specific spliceosomal protein / Pre-mRNA-processing-splicing factor 8 / U4/U6 small nuclear ribonucleoprotein Prp31 / U5 small nuclear ribonucleoprotein 40 kDa protein / Probable ATP-dependent RNA helicase DDX23 / U6 snRNA-associated Sm-like protein LSm7 / U6 snRNA-associated Sm-like protein LSm2 / U6 snRNA-associated Sm-like protein LSm5 / U6 snRNA-associated Sm-like protein LSm4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.0 Å
AuthorsAgafonov DE / Kastner B / Dybkov O / Hofele RV / Liu WT / Urlaub H / Luhrmann R / Stark H
CitationJournal: Science / Year: 2016
Title: Molecular architecture of the human U4/U6.U5 tri-snRNP.
Authors: Dmitry E Agafonov / Berthold Kastner / Olexandr Dybkov / Romina V Hofele / Wen-Ti Liu / Henning Urlaub / Reinhard Lührmann / Holger Stark /
Abstract: The U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution ...The U4/U6.U5 triple small nuclear ribonucleoprotein (tri-snRNP) is a major spliceosome building block. We obtained a three-dimensional structure of the 1.8-megadalton human tri-snRNP at a resolution of 7 angstroms using single-particle cryo-electron microscopy (cryo-EM). We fit all known high-resolution structures of tri-snRNP components into the EM density map and validated them by protein cross-linking. Our model reveals how the spatial organization of Brr2 RNA helicase prevents premature U4/U6 RNA unwinding in isolated human tri-snRNPs and how the ubiquitin C-terminal hydrolase-like protein Sad1 likely tethers the helicase Brr2 to its preactivation position. Comparison of our model with cryo-EM three-dimensional structures of the Saccharomyces cerevisiae tri-snRNP and Schizosaccharomyces pombe spliceosome indicates that Brr2 undergoes a marked conformational change during spliceosome activation, and that the scaffolding protein Prp8 is also rearranged to accommodate the spliceosome's catalytic RNA network.
History
DepositionJan 15, 2016-
Header (metadata) releaseMar 9, 2016-
Map releaseMar 9, 2016-
UpdateApr 27, 2016-
Current statusApr 27, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jcr
  • Surface level: 0.13
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6581.jpg

Downloads & links

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Map

FileDownload / File: emd_6581.map.gz / Format: CCP4 / Size: 39.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of human tri-snRNP
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 220 pix.
= 440. Å		2 Å/pix.
x 220 pix.
= 440. Å		2 Å/pix.
x 220 pix.
= 440. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13 / Movie #1: 0.13
Minimum - Maximum-0.38244939 - 0.65330416
Average (Standard dev.)0.0014018 (±0.02677352)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 440.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z440.000440.000440.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.3820.6530.001

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Supplemental data

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Sample components

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Entire : human U4/U6.U5 tri-snRNP

EntireName: human U4/U6.U5 tri-snRNP
Components
  • Sample: human U4/U6.U5 tri-snRNP
  • Protein or peptide: U4/U6.U5 tri-snRNP

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Supramolecule #1000: human U4/U6.U5 tri-snRNP

SupramoleculeName: human U4/U6.U5 tri-snRNP / type: sample / ID: 1000 / Oligomeric state: monomer / Number unique components: 1
Molecular weightExperimental: 1.8 MDa

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Macromolecule #1: U4/U6.U5 tri-snRNP

MacromoleculeName: U4/U6.U5 tri-snRNP / type: protein_or_peptide / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: Hela / Location in cell: Nucleus
Molecular weightExperimental: 1.8 MDa

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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

Concentration0.1 mg/mL
BufferpH: 7.9
Details: 20 mM HEPES, pH 7.9, 100 mM KCl, 5 mM MgCl2, 0.1 mM EDTA
GridDetails: 200 mesh copper grid with carbon support film
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateSep 10, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 4688 / Average electron dose: 45 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 74000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.0001 mm / Nominal defocus max: 5.35 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.0 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 141109

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