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- PDB-6zym: Human C Complex Spliceosome - High-resolution CORE -

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Basic information

Entry
Database: PDB / ID: 6zym
TitleHuman C Complex Spliceosome - High-resolution CORE
Components
  • (Pre-mRNA-splicing factor ...) x 5
  • 116 kDa U5 small nuclear ribonucleoprotein component
  • Cell division cycle 5-like protein
  • Corepressor interacting with RBPJ 1
  • Crooked neck-like protein 1
  • Peptidyl-prolyl cis-trans isomerase-like 1
  • Pleiotropic regulator 1
  • Pre-mRNA-processing factor 17
  • Pre-mRNA-processing-splicing factor 8
  • Protein BUD31 homolog
  • Protein FRG1
  • SNW domain-containing protein 1
  • Serine/arginine repetitive matrix protein 2
  • Spliceosome-associated protein CWC15 homolog
  • Splicing factor YJU2
  • U2 snRNAU2 spliceosomal RNA
  • U5 small nuclear ribonucleoprotein 40 kDa protein
  • U5 snRNAU5 spliceosomal RNA
  • U6 snRNAU6 spliceosomal RNA
  • WD repeat-containing protein 70
  • pre-mRNAPrimary transcript
KeywordsSPLICING / human C complex / spliceosome / hC / human step 1 spliceosome
Function / homology
Function and homology information


regulation of DNA double-strand break processing / striated muscle dense body / post-spliceosomal complex / post-mRNA release spliceosomal complex / regulation of retinoic acid receptor signaling pathway / 3'-5' RNA helicase activity / generation of catalytic spliceosome for first transesterification step / regulation of vitamin D receptor signaling pathway / embryonic brain development / pre-mRNA binding ...regulation of DNA double-strand break processing / striated muscle dense body / post-spliceosomal complex / post-mRNA release spliceosomal complex / regulation of retinoic acid receptor signaling pathway / 3'-5' RNA helicase activity / generation of catalytic spliceosome for first transesterification step / regulation of vitamin D receptor signaling pathway / embryonic brain development / pre-mRNA binding / nuclear retinoic acid receptor binding / U2-type catalytic step 1 spliceosome / RNA splicing, via transesterification reactions / Prp19 complex / positive regulation of androgen receptor activity / sno(s)RNA-containing ribonucleoprotein complex / mRNA 3'-end processing / mRNA cis splicing, via spliceosome / positive regulation of mRNA splicing, via spliceosome / U2-type spliceosomal complex / U2-type precatalytic spliceosome / regulation of mRNA splicing, via spliceosome / C2H2 zinc finger domain binding / U2-type catalytic step 2 spliceosome / positive regulation by host of viral transcription / Transport of Mature mRNA derived from an Intron-Containing Transcript / positive regulation of vitamin D receptor signaling pathway / Notch binding / nuclear vitamin D receptor binding / RNA Polymerase II Transcription Termination / Regulation of gene expression in late stage (branching morphogenesis) pancreatic bud precursor cells / muscle organ development / RUNX3 regulates NOTCH signaling / NOTCH4 Intracellular Domain Regulates Transcription / K63-linked polyubiquitin modification-dependent protein binding / microtubule organizing center / WD40-repeat domain binding / NOTCH3 Intracellular Domain Regulates Transcription / positive regulation of neurogenesis / nuclear androgen receptor binding / cyclosporin A binding / spliceosomal complex assembly / Notch-HLH transcription pathway / mRNA Splicing - Minor Pathway / Formation of paraxial mesoderm / generation of catalytic spliceosome for second transesterification step / positive regulation of transforming growth factor beta receptor signaling pathway / SMAD binding / negative regulation of DNA damage response, signal transduction by p53 class mediator / mRNA 3'-splice site recognition / protein localization to nucleus / spliceosomal tri-snRNP complex assembly / U5 snRNA binding / positive regulation of G1/S transition of mitotic cell cycle / intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / U5 snRNP / retinoic acid receptor signaling pathway / Cajal body / U2 snRNA binding / U6 snRNA binding / RNA processing / protein peptidyl-prolyl isomerization / pre-mRNA intronic binding / U1 snRNA binding / cellular response to retinoic acid / translation elongation factor activity / U4/U6 x U5 tri-snRNP complex / catalytic step 2 spliceosome / mRNA Splicing - Major Pathway / positive regulation of RNA splicing / RNA splicing / response to cocaine / nuclear receptor coactivator activity / positive regulation of protein export from nucleus / peptidylprolyl isomerase / DNA damage checkpoint signaling / peptidyl-prolyl cis-trans isomerase activity / nuclear receptor binding / Downregulation of SMAD2/3:SMAD4 transcriptional activity / RNA polymerase II transcription regulatory region sequence-specific DNA binding / spliceosomal complex / Transcription-Coupled Nucleotide Excision Repair (TC-NER) / NOTCH1 Intracellular Domain Regulates Transcription / Formation of TC-NER Pre-Incision Complex / mRNA splicing, via spliceosome / mRNA processing / fibrillar center / Z disc / Constitutive Signaling by NOTCH1 PEST Domain Mutants / Constitutive Signaling by NOTCH1 HD+PEST Domain Mutants / rRNA processing / Pre-NOTCH Transcription and Translation / nuclear matrix / Dual incision in TC-NER / positive regulation of protein import into nucleus / Gap-filling DNA repair synthesis and ligation in TC-NER / histone deacetylase binding / transcription corepressor activity / calcium-dependent protein binding / disordered domain specific binding
Similarity search - Function
Corepressor interacting with RBPJ 1 / Protein FRG1 / FRG1-like domain / CBF1-interacting co-repressor CIR, N-terminal domain / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Saf4/Yju2 protein / Splicing factor Yju2 ...Corepressor interacting with RBPJ 1 / Protein FRG1 / FRG1-like domain / CBF1-interacting co-repressor CIR, N-terminal domain / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Pre-mRNA splicing factor / N-terminal domain of CBF1 interacting co-repressor CIR / Saf4/Yju2 protein / Splicing factor Yju2 / Saf4/Yju2 protein / Actin-crosslinking / Pre-mRNA-splicing factor Isy1 / Pre-mRNA-splicing factor Isy1 superfamily / Isy1-like splicing family / : / Cyclophilin-type peptidyl-prolyl cis-trans isomerase, cyclophilin A-like / Helix hairpin bin domain superfamily / mRNA splicing factor Cwf21 domain / cwf21 domain / Pre-mRNA-processing factor 17 / : / STL11, N-terminal / WD repeat Prp46/PLRG1-like / BUD31/G10-related, conserved site / : / : / G10 protein signature 1. / G10 protein signature 2. / SKI-interacting protein SKIP, SNW domain / SKI-interacting protein, SKIP / SKIP/SNW domain / Myb-like DNA-binding domain / Pre-mRNA-splicing factor Cwf15/Cwc15 / HAT (Half-A-TPR) repeat / Cwf15/Cwc15 cell cycle control protein / Pre-mRNA-splicing factor Cwc2/Slt11 / G10 protein / Pre-mRNA-splicing factor BUD31 / Pre-mRNA splicing factor component Cdc5p/Cef1, C-terminal / pre-mRNA splicing factor component / Initiation factor eIF-4 gamma, MA3 / MA3 domain / MI domain profile. / Domain in DAP-5, eIF4G, MA-3 and other proteins. / MIF4G domain / Spt5 C-terminal domain / Spt5 C-terminal nonapeptide repeat binding Spt4 / Pre-mRNA-splicing factor Syf1-like / Middle domain of eukaryotic initiation factor 4G (eIF4G) / MIF4G-like, type 3 / Snu114, GTP-binding domain / Zinc finger, CCCH-type superfamily / 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 / : / Helicase associated domain (HA2), ratchet-like / zinc finger / DEAD-box helicase, OB fold / Oligonucleotide/oligosaccharide-binding (OB)-fold / Helicase-associated domain / Helicase associated domain (HA2), winged-helix / Helicase associated domain (HA2) Add an annotation / Myb-type HTH DNA-binding domain profile. / Zinc finger, CCCH-type / Zinc finger C3H1-type profile. / HAT (Half-A-TPR) repeat / HAT (Half-A-TPR) repeats / Myb domain / Cyclophilin-type peptidyl-prolyl cis-trans isomerase / Elongation Factor G, domain II / Elongation Factor G, domain III / DNA/RNA helicase, ATP-dependent, DEAH-box type, conserved site / DEAH-box subfamily ATP-dependent helicases signature. / Translation elongation factor EFG/EF2, domain IV / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / 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 / Cyclophilin-type peptidyl-prolyl cis-trans isomerase, conserved site / Cyclophilin-type peptidyl-prolyl cis-trans isomerase signature. / EF-G domain III/V-like / Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain profile. / Cyclophilin-type peptidyl-prolyl cis-trans isomerase domain / Cyclophilin type peptidyl-prolyl cis-trans isomerase/CLD / Cyclophilin-like domain superfamily / SANT SWI3, ADA2, N-CoR and TFIIIB'' DNA-binding domains / SANT/Myb domain / PROCT domain / Prp8 RNase domain IV, fingers region / PROCT (NUC072) domain / PRO8NT domain / PROCN domain / Pre-mRNA-processing-splicing factor 8, U6-snRNA-binding / Pre-mRNA-processing-splicing factor 8, U5-snRNA-binding / RNA recognition motif, spliceosomal PrP8 / PRP8 domain IV core
Similarity search - Domain/homology
GUANOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / : / : / RNA / RNA (> 10) / RNA (> 100) / Pleiotropic regulator 1 / Pre-mRNA-processing factor 17 / Protein BUD31 homolog ...GUANOSINE-5'-TRIPHOSPHATE / INOSITOL HEXAKISPHOSPHATE / : / : / RNA / RNA (> 10) / RNA (> 100) / Pleiotropic regulator 1 / Pre-mRNA-processing factor 17 / Protein BUD31 homolog / SNW domain-containing protein 1 / Protein FRG1 / 116 kDa U5 small nuclear ribonucleoprotein component / Pre-mRNA-processing-splicing factor 8 / Corepressor interacting with RBPJ 1 / Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16 / U5 small nuclear ribonucleoprotein 40 kDa protein / Cell division cycle 5-like protein / Splicing factor YJU2 / Crooked neck-like protein 1 / Pre-mRNA-splicing factor CWC22 homolog / Pre-mRNA-splicing factor RBM22 / WD repeat-containing protein 70 / Pre-mRNA-splicing factor CWC25 homolog / Spliceosome-associated protein CWC15 homolog / Pre-mRNA-splicing factor ISY1 homolog / Serine/arginine repetitive matrix protein 2 / Peptidyl-prolyl cis-trans isomerase-like 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsBertram, K. / Kastner, B.
Funding support Germany, 2items
OrganizationGrant numberCountry
Max Planck Society Germany
German Research Foundation (DFG)SFB 860 Germany
CitationJournal: Mol Cell / Year: 2020
Title: Structural Insights into the Roles of Metazoan-Specific Splicing Factors in the Human Step 1 Spliceosome.
Authors: Karl Bertram / Leyla El Ayoubi / Olexandr Dybkov / Dmitry E Agafonov / Cindy L Will / Klaus Hartmuth / Henning Urlaub / Berthold Kastner / Holger Stark / Reinhard Lührmann /
Abstract: Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core ...Human spliceosomes contain numerous proteins absent in yeast, whose functions remain largely unknown. Here we report a 3D cryo-EM structure of the human spliceosomal C complex at 3.4 Å core resolution and 4.5-5.7 Å at its periphery, and aided by protein crosslinking we determine its molecular architecture. Our structure provides additional insights into the spliceosome's architecture between the catalytic steps of splicing, and how proteins aid formation of the spliceosome's catalytically active RNP (ribonucleoprotein) conformation. It reveals the spatial organization of the metazoan-specific proteins PPWD1, WDR70, FRG1, and CIR1 in human C complexes, indicating they stabilize functionally important protein domains and RNA structures rearranged/repositioned during the B to C transition. Structural comparisons with human B, C, and P complexes reveal an intricate cascade of RNP rearrangements during splicing catalysis, with intermediate RNP conformations not found in yeast, and additionally elucidate the structural basis for the sequential recruitment of metazoan-specific spliceosomal proteins.
History
DepositionAug 2, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 14, 2020Provider: repository / Type: Initial release

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Assembly

Deposited unit
2: U2 snRNA
5: U5 snRNA
6: U6 snRNA
9: Corepressor interacting with RBPJ 1
A: Pre-mRNA-processing-splicing factor 8
B: 116 kDa U5 small nuclear ribonucleoprotein component
C: SNW domain-containing protein 1
D: Pleiotropic regulator 1
E: Pre-mRNA-processing factor 17
F: U5 small nuclear ribonucleoprotein 40 kDa protein
L: Cell division cycle 5-like protein
O: Crooked neck-like protein 1
P: Pre-mRNA-splicing factor RBM22
Q: Protein BUD31 homolog
R: Spliceosome-associated protein CWC15 homolog
S: Serine/arginine repetitive matrix protein 2
T: Pre-mRNA-splicing factor CWC22 homolog
V: Peptidyl-prolyl cis-trans isomerase-like 1
Y: pre-mRNA
Z: pre-mRNA
p: WD repeat-containing protein 70
r: Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16
s: Pre-mRNA-splicing factor ISY1 homolog
t: Pre-mRNA-splicing factor CWC25 homolog
u: Splicing factor YJU2
x: Protein FRG1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,956,92940
Polymers1,955,16626
Non-polymers1,76314
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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RNA chain , 4 types, 5 molecules 256YZ

#1: 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) / References: GenBank: 36516
#2: RNA chain U5 snRNA / U5 spliceosomal RNA


Mass: 36908.668 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: GenBank: 36515
#3: RNA chain U6 snRNA / U6 spliceosomal RNA


Mass: 25480.240 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela S3
#19: RNA chain pre-mRNA / Primary transcript


Mass: 103979.148 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: Hela S3

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Protein , 16 types, 16 molecules 9ABCDEFLOQRSVpux

#4: Protein Corepressor interacting with RBPJ 1 / CBF1-interacting corepressor / Recepin


Mass: 52441.750 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q86X95
#5: Protein Pre-mRNA-processing-splicing factor 8 / 220 kDa U5 snRNP-specific protein / PRP8 homolog / Splicing factor Prp8 / p220


Mass: 207470.719 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q6P2Q9
#6: Protein 116 kDa U5 small nuclear ribonucleoprotein component / Elongation factor Tu GTP-binding domain-containing protein 2 / SNU114 homolog / hSNU114 / U5 snRNP- ...Elongation factor Tu GTP-binding domain-containing protein 2 / SNU114 homolog / hSNU114 / U5 snRNP-specific protein / 116 kDa / U5-116 kDa


Mass: 107225.898 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q15029
#7: Protein SNW domain-containing protein 1 / Nuclear protein SkiP / Nuclear receptor coactivator NCoA-62 / Ski-interacting protein


Mass: 61610.703 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q13573
#8: Protein Pleiotropic regulator 1


Mass: 57280.758 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O43660
#9: Protein Pre-mRNA-processing factor 17 / Cell division cycle 40 homolog / EH-binding protein 3 / Ehb3 / PRP17 homolog / hPRP17


Mass: 65612.180 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: O60508
#10: Protein U5 small nuclear ribonucleoprotein 40 kDa protein / U5-40K / 38 kDa-splicing factor / Prp8-binding protein / hPRP8BP / U5 snRNP-specific 40 kDa protein ...U5-40K / 38 kDa-splicing factor / Prp8-binding protein / hPRP8BP / U5 snRNP-specific 40 kDa protein / WD repeat-containing protein 57


Mass: 39359.492 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q96DI7
#11: Protein Cell division cycle 5-like protein / Cdc5-like protein / Pombe cdc5-related protein


Mass: 92406.883 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q99459
#12: Protein Crooked neck-like protein 1 / Crooked neck homolog / hCrn


Mass: 100610.008 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9BZJ0
#14: Protein Protein BUD31 homolog / Protein EDG-2 / Protein G10 homolog


Mass: 17032.850 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: P41223
#15: Protein Spliceosome-associated protein CWC15 homolog


Mass: 26674.447 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9P013
#16: Protein Serine/arginine repetitive matrix protein 2 / 300 kDa nuclear matrix antigen / Serine/arginine-rich splicing factor-related nuclear matrix ...300 kDa nuclear matrix antigen / Serine/arginine-rich splicing factor-related nuclear matrix protein of 300 kDa / Ser/Arg-related nuclear matrix protein of 300 kDa / Splicing coactivator subunit SRm300 / Tax-responsive enhancer element-binding protein 803 / TaxREB803


Mass: 300255.312 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9UQ35
#18: Protein Peptidyl-prolyl cis-trans isomerase-like 1 / PPIase / Rotamase PPIL1


Mass: 18257.805 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9Y3C6, peptidylprolyl isomerase
#20: Protein WD repeat-containing protein 70


Mass: 73306.172 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9NW82
#24: Protein Splicing factor YJU2 / Coiled-coil domain-containing protein 94


Mass: 21452.719 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9BW85
#25: Protein Protein FRG1 / FSHD region gene 1 protein


Mass: 29227.492 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q14331

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Pre-mRNA-splicing factor ... , 5 types, 5 molecules PTrst

#13: Protein Pre-mRNA-splicing factor RBM22 / RNA-binding motif protein 22 / Zinc finger CCCH domain-containing protein 16


Mass: 25223.928 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9NW64
#17: Protein Pre-mRNA-splicing factor CWC22 homolog / Nucampholin homolog / fSAPb


Mass: 105646.578 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9HCG8
#21: Protein Pre-mRNA-splicing factor ATP-dependent RNA helicase PRP16 / ATP-dependent RNA helicase DHX38 / DEAH box protein 38


Mass: 140720.641 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q92620, RNA helicase
#22: Protein Pre-mRNA-splicing factor ISY1 homolog


Mass: 33046.254 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9ULR0
#23: Protein Pre-mRNA-splicing factor CWC25 homolog / Coiled-coil domain-containing protein 49 / Spliceosome-associated protein homolog CWC25


Mass: 49769.938 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / References: UniProt: Q9NXE8

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Non-polymers , 4 types, 14 molecules

#26: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
#27: Chemical ChemComp-IHP / INOSITOL HEXAKISPHOSPHATE / MYO-INOSITOL HEXAKISPHOSPHATE / INOSITOL 1,2,3,4,5,6-HEXAKISPHOSPHATE / Phytic acid


Mass: 660.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H18O24P6
#28: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE / Guanosine triphosphate


Mass: 523.180 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Comment: GTP, energy-carrying molecule*YM
#29: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Zn

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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

ComponentName: Human C complex Spliceosome - High resolution core / Type: COMPLEX / Entity ID: #1-#25 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa S3
Buffer solutionpH: 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES-KOH, pH 7.9HEPES-KOH1
21.5 mMMagnesium ChlorideMgCl21
3150 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse particle distribution
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK II / 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 132000 X / Nominal defocus max: -3 nm / Nominal defocus min: -1 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 120 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 28279
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1751359
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 411539 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01149836
ELECTRON MICROSCOPYf_angle_d1.268647
ELECTRON MICROSCOPYf_dihedral_angle_d22.1388767
ELECTRON MICROSCOPYf_chiral_restr0.0837701
ELECTRON MICROSCOPYf_plane_restr0.0087966

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