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- PDB-5gap: Body region of the U4/U6.U5 tri-snRNP -

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Entry
Database: PDB / ID: 5gap
TitleBody region of the U4/U6.U5 tri-snRNP
Components
  • (Pre-mRNA-splicing factor ...) x 2
  • (U4/U6 small nuclear ribonucleoprotein ...) x 2
  • 13 kDa ribonucleoprotein-associated protein
  • Pre-mRNA-processing factor 31
  • Pre-mRNA-splicing helicase BRR2
  • Spliceosomal protein DIB1Spliceosome
  • U4 snRNA, 5' region, nucleotides 1-67U4 spliceosomal RNA
  • U5 snRNAU5 spliceosomal RNA
  • U6 snRNAU6 spliceosomal RNA
  • unknown protein
KeywordsTRANSCRIPTION / snRNP / spliceosome / RNA-protein complex / U4/U6.U5 snRNP
Function / homology
Function and homology information


spliceosomal conformational changes to generate catalytic conformation / U4/U6 snRNP / snoRNA splicing / rRNA 2'-O-methylation / positive regulation of RNA binding / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / box C/D RNP complex / U4 snRNP / spliceosomal tri-snRNP complex / U4 snRNA binding ...spliceosomal conformational changes to generate catalytic conformation / U4/U6 snRNP / snoRNA splicing / rRNA 2'-O-methylation / positive regulation of RNA binding / spliceosome conformational change to release U4 (or U4atac) and U1 (or U11) / box C/D RNP complex / U4 snRNP / spliceosomal tri-snRNP complex / U4 snRNA binding / U3 snoRNA binding / generation of catalytic spliceosome for second transesterification step / spliceosomal snRNP assembly / mRNA 3'-splice site recognition / mRNA 5'-splice site recognition / precatalytic spliceosome / spliceosomal tri-snRNP complex assembly / rRNA methylation / U5 snRNP / U5 snRNA binding / U2 snRNA binding / U6 snRNA binding / pre-mRNA intronic binding / U4/U6 x U5 tri-snRNP complex / maturation of SSU-rRNA / U1 snRNA binding / small-subunit processome / isopeptidase activity / catalytic step 2 spliceosome / maturation of LSU-rRNA / spliceosomal complex / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / metallopeptidase activity / mRNA splicing, via spliceosome / RNA helicase / RNA helicase activity / mRNA binding / nucleolus / mitochondrion / RNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus
PROCT domain / PROCN domain / WD40-repeat-containing domain / WD40/YVTN repeat-like-containing domain superfamily / Pre-mRNA processing factor 4 (PRP4)-like / Immunoglobulin E-set / Helicase superfamily 1/2, ATP-binding domain / Pre-mRNA-splicing factor 3 / Tetratricopeptide repeat-containing domain / NOSIC ...PROCT domain / PROCN domain / WD40-repeat-containing domain / WD40/YVTN repeat-like-containing domain superfamily / Pre-mRNA processing factor 4 (PRP4)-like / Immunoglobulin E-set / Helicase superfamily 1/2, ATP-binding domain / Pre-mRNA-splicing factor 3 / Tetratricopeptide repeat-containing domain / NOSIC / PRO8NT domain / Prp31 C-terminal / Ribonuclease H-like superfamily / Tetratricopeptide-like helical domain superfamily / DEAD/DEAH box helicase domain / Domain of unknown function DUF1115 / PRP1 splicing factor, N-terminal / Sec63 domain / Dim1 family / Ribosomal protein L7Ae/L30e/S12e/Gadd45 / Ribosomal protein L7Ae conserved site / Ribosomal protein L7Ae/L8/Nhp2 family / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / Nop domain / 50S ribosomal protein L30e-like / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding domain superfamily / Nop, C-terminal domain / Brr2, N-terminal helicase PWI domain / MPN domain / Winged helix DNA-binding domain superfamily / WD40-repeat-containing domain superfamily / Thioredoxin-like superfamily / Nop domain superfamily / C2 domain superfamily / Pre-mRNA-processing-splicing factor 8 / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / P-loop containing nucleoside triphosphate hydrolase / Pre-mRNA-processing factor 6/Prp1/STA1 / U4/U6 small nuclear ribonucleoprotein Prp31 / U4/U6 small nuclear ribonucleoprotein Prp3 / PRP8 domain IV core / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Tetratricopeptide repeat / RNA recognition motif, spliceosomal PrP8 / HAT (Half-A-TPR) repeat / H/ACA ribonucleoprotein complex, subunit Nhp2, eukaryote / Prp8 RNase domain IV, fingers region / WD40 repeat / Ribosomal protein L7Ae/L30e/S12e/Gadd45 family / PRP1 splicing factor, N-terminal / WD domain, G-beta repeat / pre-mRNA processing factor 3 (PRP3) / Protein of unknown function (DUF1115) / Prp31 C terminal domain / Mitosis protein DIM1 / N-terminal helicase PWI domain / Sec63 Brl domain / Helicase conserved C-terminal domain / Prp8 RNase domain IV, palm region / JAB1/MPN/MOV34 metalloenzyme domain / PRP8 domain IV core / Helicase, C-terminal / Ribosomal protein L30/S12 / 60s Ribosomal Protein L30; Chain: A; / Glutaredoxin / Glutaredoxin / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
gb:807071964: / Pre-mRNA-splicing factor 8 / Pre-mRNA-splicing helicase BRR2 / U4/U6 small nuclear ribonucleoprotein PRP3 / 13 kDa ribonucleoprotein-associated protein / Pre-mRNA-processing factor 31 / gb:807071959: / Spliceosomal protein DIB1 / U4/U6 small nuclear ribonucleoprotein PRP4 / gb:807071957: / Pre-mRNA-splicing factor 6
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsNguyen, T.H.D. / Galej, W.P. / Oubridge, C. / Bai, X.C. / Newman, A. / Scheres, S. / Nagai, K.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom) United Kingdom
CitationJournal: Nature / Year: 2016
Title: Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution.
Authors: Thi Hoang Duong Nguyen / Wojciech P Galej / Xiao-Chen Bai / Chris Oubridge / Andrew J Newman / Sjors H W Scheres / Kiyoshi Nagai /
Abstract: U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led ...U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5'-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre.
Validation Report
SummaryFull reportAbout validation report
History
DepositionDec 15, 2015Deposition site: RCSB / Processing site: PDBE
Revision 1.0Jan 27, 2016Provider: repository / Type: Initial release
Revision 1.1Mar 2, 2016Group: Database references
Revision 1.2Aug 30, 2017Group: Author supporting evidence / Data collection / Derived calculations
Category: em_imaging_optics / em_software ...em_imaging_optics / em_software / pdbx_audit_support / struct_conn
Item: _em_imaging_optics.energyfilter_name / _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.3Oct 2, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.length_a / _cell.length_b / _cell.length_c

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Structure visualization

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Assembly

Deposited unit
V: U4 snRNA, 5' region, nucleotides 1-67
W: U6 snRNA
U: U5 snRNA
x: unknown protein
A: Pre-mRNA-splicing factor 8
H: U4/U6 small nuclear ribonucleoprotein PRP4
J: Pre-mRNA-splicing factor 6
D: Spliceosomal protein DIB1
F: Pre-mRNA-processing factor 31
G: U4/U6 small nuclear ribonucleoprotein PRP3
K: 13 kDa ribonucleoprotein-associated protein
B: Pre-mRNA-splicing helicase BRR2


Theoretical massNumber of molelcules
Total (without water)959,00512
Polymers959,00512
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area60560 Å2
ΔGint-428 kcal/mol
Surface area169960 Å2
MethodPISA

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Components

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RNA chain , 3 types, 3 molecules VWU

#1: RNA chain U4 snRNA, 5' region, nucleotides 1-67 / U4 spliceosomal RNA


Mass: 21528.688 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: U4 snRNA 5' region, nucleotides 1-67. / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: GenBank: 807071957
#2: RNA chain U6 snRNA / U6 spliceosomal RNA


Mass: 35883.176 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: GenBank: 807071964
#3: RNA chain U5 snRNA / U5 spliceosomal RNA


Mass: 68643.344 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: GenBank: 807071959

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Protein , 5 types, 5 molecules xDFKB

#4: Protein unknown protein


Mass: 6996.616 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: Clear helical regions were built as poly(Ala) but could not be assigned to a specific protein.
Source: (natural) Saccharomyces cerevisiae (baker's yeast)
#8: Protein Spliceosomal protein DIB1 / Spliceosome / Dib1


Mass: 16798.387 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q06819
#9: Protein Pre-mRNA-processing factor 31 / Prp31


Mass: 56382.516 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P49704
#11: Protein 13 kDa ribonucleoprotein-associated protein / Small nuclear ribonucleoprotein-associated protein 1 / Snu13


Mass: 13582.855 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P39990
#12: Protein Pre-mRNA-splicing helicase BRR2 / Protein Snu246


Mass: 246470.266 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P32639, RNA helicase

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Pre-mRNA-splicing factor ... , 2 types, 2 molecules AJ

#5: Protein Pre-mRNA-splicing factor 8 / Prp8


Mass: 279867.469 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P33334
#7: Protein Pre-mRNA-splicing factor 6 / Prp6


Mass: 104370.133 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P19735

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U4/U6 small nuclear ribonucleoprotein ... , 2 types, 2 molecules HG

#6: Protein U4/U6 small nuclear ribonucleoprotein PRP4 / Pre-mRNA-processing protein 4 / Prp4


Mass: 52506.984 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P20053
#10: Protein U4/U6 small nuclear ribonucleoprotein PRP3 / Pre-mRNA-splicing factor 3 / Prp3


Mass: 55974.320 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q03338

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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: Body region of the U4/U6.U5 tri-snRNP complex / Type: COMPLEX / Entity ID: #1-#12 / Source: NATURAL
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 7.9
Buffer componentConc.: 1 mM / Name: DTT
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 3.5 microlitre of sample was applied to grid.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Grids were blotted at 4 deg C for 2 seconds before plunging.

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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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Calibrated magnification: 35714 X / Nominal defocus max: 3500 nm / Nominal defocus min: 500 nm / Cs: 2 mm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 16 sec. / Electron dose: 38 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 2477
EM imaging opticsEnergyfilter name: GIF Quantum
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: REFMAC / Version: 5.8.0124 / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION1.4particle selection
4CTFFIND4CTF correction
7Coot0.8.2model fitting
9REFMAC5.8model refinement316
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 473827
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 140155 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 3.6→180.18 Å / Cor.coef. Fo:Fc: 0.975 / SU B: 24.876 / SU ML: 0.368 / ESU R: 0.688
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.27766 --
Obs0.27766 192905 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 316.59 Å2
Baniso -1Baniso -2Baniso -3
1--0.55 Å22.6 Å2-0.83 Å2
2--3.27 Å2-0.36 Å2
3----2.73 Å2
Refinement stepCycle: 1 / Total: 31573
Refine LS restraints
Refinement-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01932617
ELECTRON MICROSCOPYr_bond_other_d0.0020.0229370
ELECTRON MICROSCOPYr_angle_refined_deg1.3611.88444645
ELECTRON MICROSCOPYr_angle_other_deg0.982367630
ELECTRON MICROSCOPYr_dihedral_angle_1_deg11.1885.5053966
ELECTRON MICROSCOPYr_dihedral_angle_2_deg35.06524.1541324
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.644155219
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.0115193
ELECTRON MICROSCOPYr_chiral_restr0.2160.2055069
ELECTRON MICROSCOPYr_gen_planes_refined0.0050.0234318
ELECTRON MICROSCOPYr_gen_planes_other0.0020.027415
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it15.6230.82914323
ELECTRON MICROSCOPYr_mcbond_other15.62130.82814322
ELECTRON MICROSCOPYr_mcangle_it24.07246.27817874
ELECTRON MICROSCOPYr_mcangle_other24.07146.27917875
ELECTRON MICROSCOPYr_scbond_it17.42232.70418294
ELECTRON MICROSCOPYr_scbond_other17.42232.70518295
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other29.08248.3326772
ELECTRON MICROSCOPYr_long_range_B_refined36.26566311
ELECTRON MICROSCOPYr_long_range_B_other36.26466312
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.6→3.693 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork1.415 14158 -
Rfree-0 -
Obs--100 %

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