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- PDB-7q3l: Human 17S U2 snRNP 5' domain -

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Basic information

Entry
Database: PDB / ID: 7q3l
TitleHuman 17S U2 snRNP 5' domain
Components
  • (Splicing factor 3B subunit ...) x 4
  • HIV Tat-specific factor 1
  • PHD finger-like domain-containing protein 5A
  • Probable ATP-dependent RNA helicase DDX46
  • Splicing factor 3A subunit 3
  • U2 snRNAU2 spliceosomal RNA
KeywordsNUCLEAR PROTEIN / snRNP / Spliceosome / U2 snRNP / Splicing
Function / homology
Function and homology information


U11/U12 snRNP / chromatin-protein adaptor activity / B-WICH complex / splicing factor binding / protein localization to site of double-strand break / U12-type spliceosomal complex / RNA splicing, via transesterification reactions / poly-ADP-D-ribose modification-dependent protein binding / : / U2-type spliceosomal complex ...U11/U12 snRNP / chromatin-protein adaptor activity / B-WICH complex / splicing factor binding / protein localization to site of double-strand break / U12-type spliceosomal complex / RNA splicing, via transesterification reactions / poly-ADP-D-ribose modification-dependent protein binding / : / U2-type spliceosomal complex / U2-type precatalytic spliceosome / U2-type prespliceosome assembly / U2 snRNP / SAGA complex / positive regulation of transcription by RNA polymerase III / U2-type prespliceosome / precatalytic spliceosome / spliceosomal complex assembly / positive regulation of transcription by RNA polymerase I / mRNA Splicing - Minor Pathway / regulation of RNA splicing / mRNA 3'-splice site recognition / regulation of DNA repair / Cajal body / U2 snRNA binding / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / RNA splicing / stem cell differentiation / double-strand break repair via homologous recombination / spliceosomal complex / B-WICH complex positively regulates rRNA expression / negative regulation of protein catabolic process / mRNA splicing, via spliceosome / fibrillar center / mRNA processing / nuclear matrix / site of double-strand break / RNA helicase activity / RNA helicase / nuclear speck / chromatin remodeling / mRNA binding / protein-containing complex binding / nucleolus / positive regulation of DNA-templated transcription / ATP hydrolysis activity / positive regulation of transcription by RNA polymerase II / DNA binding / RNA binding / zinc ion binding / nucleoplasm / ATP binding / nucleus
Similarity search - Function
TatSF1-like, RNA recognition motif 1 / TatSF1-like / Splicing factor SF3a60 binding domain / Splicing factor SF3a60 binding domain / : / Replication stress response SDE2 C-terminal / Splicing factor SF3a60 /Prp9 subunit, C-terminal / SF3A3 domain / SF3a60/Prp9 C-terminal / Pre-mRNA-splicing factor SF3A3, of SF3a complex, Prp9 ...TatSF1-like, RNA recognition motif 1 / TatSF1-like / Splicing factor SF3a60 binding domain / Splicing factor SF3a60 binding domain / : / Replication stress response SDE2 C-terminal / Splicing factor SF3a60 /Prp9 subunit, C-terminal / SF3A3 domain / SF3a60/Prp9 C-terminal / Pre-mRNA-splicing factor SF3A3, of SF3a complex, Prp9 / Splicing factor 3B, subunit 5 / Splicing factor 3B subunit 1 / Splicing factor 3B subunit 1 / Domain of unknown function DUF382 / Domain of unknown function (DUF382) / PHF5-like / PHF5-like protein / PSP, proline-rich / PSP / proline-rich domain in spliceosome associated proteins / Splicing factor 3B subunit 5/RDS3 complex subunit 10 / Splicing factor 3B subunit 10 (SF3b10) / Matrin/U1-C, C2H2-type zinc finger / Splicing factor 3B subunit 1-like / Zinc finger matrin-type profile. / SAP domain / SAP motif profile. / Putative DNA-binding (bihelical) motif predicted to be involved in chromosomal organisation / SAP domain / Cleavage/polyadenylation specificity factor, A subunit, N-terminal / Mono-functional DNA-alkylating methyl methanesulfonate N-term / Cleavage/polyadenylation specificity factor, A subunit, C-terminal / CPSF A subunit region / DEAD-box subfamily ATP-dependent helicases signature. / ATP-dependent RNA helicase DEAD-box, conserved site / RNA helicase, DEAD-box type, Q motif / DEAD-box RNA helicase Q motif profile. / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / Helicase conserved C-terminal domain / RNA-binding domain superfamily / Armadillo-like helical / 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 / Armadillo-type fold / Nucleotide-binding alpha-beta plait domain superfamily / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / 17S U2 SnRNP complex component HTATSF1 / Splicing factor 3B subunit 1 / Splicing factor 3A subunit 3 / Splicing factor 3B subunit 2 / Splicing factor 3B subunit 3 / Probable ATP-dependent RNA helicase DDX46 ...: / RNA / RNA (> 10) / RNA (> 100) / 17S U2 SnRNP complex component HTATSF1 / Splicing factor 3B subunit 1 / Splicing factor 3A subunit 3 / Splicing factor 3B subunit 2 / Splicing factor 3B subunit 3 / Probable ATP-dependent RNA helicase DDX46 / PHD finger-like domain-containing protein 5A / Splicing factor 3B subunit 5
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.21 Å
AuthorsTholen, J. / Galej, W.P.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)950278European Union
CitationJournal: Science / Year: 2022
Title: Structural basis of branch site recognition by the human spliceosome.
Authors: Jonas Tholen / Michal Razew / Felix Weis / Wojciech P Galej /
Abstract: Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and ...Recognition of the intron branch site (BS) by the U2 small nuclear ribonucleoprotein (snRNP) is a critical event during spliceosome assembly. In mammals, BS sequences are poorly conserved, and unambiguous intron recognition cannot be achieved solely through a base-pairing mechanism. We isolated human 17 U2 snRNP and reconstituted in vitro its adenosine 5´-triphosphate (ATP)–dependent remodeling and binding to the pre–messenger RNA substrate. We determined a series of high-resolution (2.0 to 2.2 angstrom) structures providing snapshots of the BS selection process. The substrate-bound U2 snRNP shows that SF3B6 stabilizes the BS:U2 snRNA duplex, which could aid binding of introns with poor sequence complementarity. ATP-dependent remodeling uncoupled from substrate binding captures U2 snRNA in a conformation that competes with BS recognition, providing a selection mechanism based on branch helix stability.
History
DepositionOct 28, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 30, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Splicing factor 3B subunit 2
C: Splicing factor 3B subunit 3
E: Splicing factor 3B subunit 5
G: PHD finger-like domain-containing protein 5A
2: U2 snRNA
9: Splicing factor 3A subunit 3
q: HIV Tat-specific factor 1
A: Splicing factor 3B subunit 1
p: Probable ATP-dependent RNA helicase DDX46
hetero molecules


Theoretical massNumber of molelcules
Total (without water)732,54613
Polymers732,2859
Non-polymers2624
Water3,243180
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area30750 Å2
ΔGint-114 kcal/mol
Surface area99330 Å2

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Components

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Splicing factor 3B subunit ... , 4 types, 4 molecules BCEA

#1: Protein Splicing factor 3B subunit 2 / Pre-mRNA-splicing factor SF3b 145 kDa subunit / SF3b145 / Spliceosome-associated protein 145 / SAP 145


Mass: 100377.812 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q13435
#2: Protein Splicing factor 3B subunit 3 / Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein ...Pre-mRNA-splicing factor SF3b 130 kDa subunit / SF3b130 / STAF130 / Spliceosome-associated protein 130 / SAP 130


Mass: 135718.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15393
#3: Protein Splicing factor 3B subunit 5 / SF3b5 / Pre-mRNA-splicing factor SF3b 10 kDa subunit


Mass: 10149.369 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: Q9BWJ5
#8: Protein Splicing factor 3B subunit 1 / Pre-mRNA-splicing factor SF3b 155 kDa subunit / SF3b155 / Spliceosome-associated protein 155 / SAP 155


Mass: 146024.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: O75533

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Protein , 4 types, 4 molecules G9qp

#4: Protein PHD finger-like domain-containing protein 5A / PHD finger-like domain protein 5A / Splicing factor 3B-associated 14 kDa protein / SF3b14b


Mass: 12427.524 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: Q7RTV0
#6: Protein Splicing factor 3A subunit 3 / SF3a60 / Spliceosome-associated protein 61 / SAP 61


Mass: 58934.844 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: Q12874
#7: Protein HIV Tat-specific factor 1 / Tat-SF1


Mass: 90888.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Cell line: HEK293F / Gene: HTATSF1 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: O43719
#9: Protein Probable ATP-dependent RNA helicase DDX46 / DEAD box protein 46 / PRP5 homolog


Mass: 117576.484 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: UniProt: Q7L014, RNA helicase

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RNA chain , 1 types, 1 molecules 2

#5: RNA chain U2 snRNA / U2 spliceosomal RNA


Mass: 60186.445 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: HEK293F / References: GenBank: NR_002716

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Non-polymers , 2 types, 184 molecules

#10: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#11: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 180 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
117S U2 snRNPCOMPLEX17S U2 snRNP isolated from nuclear extract of HEK293F cells by affinity chromatography.#1-#90MULTIPLE SOURCES
217S U2 snRNPCOMPLEX#1-#6, #8-#91NATURAL
3HIV Tat-specific factor 1COMPLEX#71RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
111.08 MDaNO
210.86 MDaNO
310.3 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.9
Details: Sample after desalting may have also contained up to 5% glycerol.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMPotassium chlorideKCl1
32 mMMagnesium chlorideMgCl21
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 53.45 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 15531
Image scansWidth: 5760 / Height: 4092

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4CTFFIND4.1.14CTF correction
7Coot0.9.4.1model fitting
8UCSF ChimeraX1.1model fitting
9ISOLDE1.1model fitting
11REFMAC5model refinement
12LIBGmodel refinement
13cryoSPARC2.15initial Euler assignmentAb-initio reconstruction
14RELION3.1final Euler assignment
15RELION3.1classification
16RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1500165
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.21 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 225934 / Symmetry type: POINT
Atomic model buildingSpace: RECIPROCAL
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
16EN4

6en4

A1491-1304
26Y5Q

6y5q

B1452-598
36EN4

6en4

C11-1217
46EN4

6en4

E112-81
56EN4

6en4

G18-90
66Y5Q

6y5q

91392-493
RefinementResolution: 2.3→144.64 Å / Cor.coef. Fo:Fc: 0.884 / SU B: 6.568 / SU ML: 0.149 / ESU R: 0.21
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.35289 --
obs0.35289 299215 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 108.751 Å2
Baniso -1Baniso -2Baniso -3
1-1.45 Å22.83 Å2-0.05 Å2
2--0.32 Å20.41 Å2
3----1.77 Å2
Refinement stepCycle: 1 / Total: 15856
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0050.01216109
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.4651.6521984
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.4995.5392050
ELECTRON MICROSCOPYr_dihedral_angle_2_deg34.27122.289817
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.581152596
ELECTRON MICROSCOPYr_dihedral_angle_4_deg19.71615101
ELECTRON MICROSCOPYr_chiral_restr0.1180.2012152
ELECTRON MICROSCOPYr_gen_planes_refined0.010.0211890
ELECTRON MICROSCOPYr_gen_planes_other
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_it1.24410.1647428
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it2.35615.2139250
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it0.74311.7428681
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined12.85870139
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 2.3→2.36 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.963 22216 -
obs--100 %

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