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- PDB-7b7u: Cryo-EM structure of mammalian RNA polymerase II in complex with ... -

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Entry
Database: PDB / ID: 7b7u
TitleCryo-EM structure of mammalian RNA polymerase II in complex with human RPAP2
Components
  • (DNA-directed RNA polymerase ...) x 6
  • DNA-directed RNA polymerases I, II, and III subunit RPABC3
  • Putative RNA polymerase II subunit B1 CTD phosphatase RPAP2
  • RNA polymerase II subunit K
  • RNA_pol_L_2 domain-containing protein
  • Uncharacterized Protein
  • Unknown
KeywordsTRANSCRIPTION / RPAP2 / RNA polymerase II / Nuclear import
Function / homology
Function and homology information


snRNA transcription / RNA polymerase II CTD heptapeptide repeat phosphatase activity / RNA polymerase core enzyme binding / PERK-mediated unfolded protein response / B-WICH complex positively regulates rRNA expression / RNA Polymerase I Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase I Transcription Termination / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase III Transcription Initiation From Type 2 Promoter ...snRNA transcription / RNA polymerase II CTD heptapeptide repeat phosphatase activity / RNA polymerase core enzyme binding / PERK-mediated unfolded protein response / B-WICH complex positively regulates rRNA expression / RNA Polymerase I Transcription Initiation / RNA Polymerase I Promoter Escape / RNA Polymerase I Transcription Termination / RNA Polymerase III Transcription Initiation From Type 1 Promoter / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Polymerase III Transcription Initiation From Type 3 Promoter / Formation of RNA Pol II elongation complex / Formation of the Early Elongation Complex / Transcriptional regulation by small RNAs / RNA Polymerase II Pre-transcription Events / TP53 Regulates Transcription of DNA Repair Genes / FGFR2 alternative splicing / RNA polymerase II transcribes snRNA genes / mRNA Capping / mRNA Splicing - Minor Pathway / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Elongation / RNA Polymerase II Transcription Initiation And Promoter Clearance / RNA Pol II CTD phosphorylation and interaction with CE / Estrogen-dependent gene expression / Formation of TC-NER Pre-Incision Complex / Dual incision in TC-NER / Gap-filling DNA repair synthesis and ligation in TC-NER / mRNA Splicing - Major Pathway / transcription preinitiation complex / protein serine/threonine phosphatase activity / myosin phosphatase activity / protein-serine/threonine phosphatase / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / RNA polymerase II transcribes snRNA genes / RNA polymerase II activity / transcription elongation by RNA polymerase I / transcription-coupled nucleotide-excision repair / tRNA transcription by RNA polymerase III / RNA polymerase I complex / RNA polymerase III complex / RNA polymerase II, core complex / core promoter sequence-specific DNA binding / transcription initiation at RNA polymerase II promoter / euchromatin / ribonucleoside binding / fibrillar center / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / transcription by RNA polymerase II / nucleic acid binding / protein dimerization activity / DNA-templated transcription / nucleolus / DNA binding / zinc ion binding / nucleoplasm / nucleus / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Rtr1/RPAP2 domain / Rtr1/RPAP2 domain superfamily / Rtr1/RPAP2 / Rtr1/RPAP2 family / RTR1-type zinc finger. / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. ...Rtr1/RPAP2 domain / Rtr1/RPAP2 domain superfamily / Rtr1/RPAP2 / Rtr1/RPAP2 family / RTR1-type zinc finger. / RNA polymerase II, heptapeptide repeat, eukaryotic / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1 C-terminal repeat / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily / RNA polymerase Rpb1, domain 7 / Pol II subunit B9, C-terminal zinc ribbon / RNA polymerase RBP11 / Zinc finger TFIIS-type signature. / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 5 / RNA polymerase Rpb2, domain 4 / RNA polymerase Rpb2, domain 4 / DNA-directed RNA polymerase, M/15kDa subunit / RNA polymerases M/15 Kd subunit / RNA polymerase subunit 9 / DNA-directed RNA polymerase M, 15kDa subunit, conserved site / RNA polymerases M / 15 Kd subunits signature. / DNA-directed RNA polymerase subunit/transcription factor S / : / RNA polymerase, Rpb8 / DNA-directed RNA polymerases I, II, and III subunit RPABC4 / RNA polymerase Rpb8 / RNA polymerase subunit 8 / RNA polymerase, Rpb5, N-terminal / RNA polymerase Rpb5, N-terminal domain superfamily / RNA polymerase Rpb5, N-terminal domain / DNA-directed RNA polymerase, subunit RPB6 / DNA directed RNA polymerase, 7 kDa subunit / RNA polymerase archaeal subunit P/eukaryotic subunit RPABC4 / RNA polymerase, subunit H/Rpb5, conserved site / RNA polymerases H / 23 Kd subunits signature. / RNA polymerase subunit CX / DNA-directed RNA polymerase, 30-40kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo3/Rpb3/RPAC1 / RNA polymerases D / 30 to 40 Kd subunits signature. / DNA-directed RNA polymerase Rpb11, 13-16kDa subunit, conserved site / DNA-directed RNA polymerase subunit Rpo11 / RNA polymerases L / 13 to 16 Kd subunits signature. / Zinc finger, TFIIS-type / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / RPB5-like RNA polymerase subunit superfamily / RNA polymerase Rpb5, C-terminal domain / Archaeal Rpo6/eukaryotic RPB6 RNA polymerase subunit / DNA-directed RNA polymerase, 14-18kDa subunit, conserved site / RNA polymerases K / 14 to 18 Kd subunits signature. / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / RNA polymerase Rpb1, clamp domain superfamily / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 4 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain
Similarity search - Domain/homology
DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase II subunit RPB11-a / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase subunit / RNA polymerase II, I and III subunit K / DNA-directed RNA polymerase II subunit E / DNA-directed RNA polymerase II subunit RPB9 / Putative RNA polymerase II subunit B1 CTD phosphatase RPAP2
Similarity search - Component
Biological speciesHomo sapiens (human)
Sus scrofa domesticus (domestic pig)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.8 Å
AuthorsFianu, I. / Dienemann, C. / Aibara, S. / Schilbach, S. / Cramer, P.
Funding support Germany, 3items
OrganizationGrant numberCountry
European Research Council (ERC)693023 Germany
German Research Foundation (DFG)SFB860, SPP2191, EXC 2067/1-390729940 Germany
Marie Sklodowska-Curie Actions, FragNET ITN894862 Germany
CitationJournal: Commun Biol / Year: 2021
Title: Cryo-EM structure of mammalian RNA polymerase II in complex with human RPAP2.
Authors: Isaac Fianu / Christian Dienemann / Shintaro Aibara / Sandra Schilbach / Patrick Cramer /
Abstract: Nuclear import of RNA polymerase II (Pol II) involves the conserved factor RPAP2. Here we report the cryo-electron microscopy (cryo-EM) structure of mammalian Pol II in complex with human RPAP2 at 2. ...Nuclear import of RNA polymerase II (Pol II) involves the conserved factor RPAP2. Here we report the cryo-electron microscopy (cryo-EM) structure of mammalian Pol II in complex with human RPAP2 at 2.8 Å resolution. The structure shows that RPAP2 binds between the jaw domains of the polymerase subunits RPB1 and RPB5. RPAP2 is incompatible with binding of downstream DNA during transcription and is displaced upon formation of a transcription pre-initiation complex.
History
DepositionDec 11, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 19, 2021Provider: repository / Type: Initial release
Revision 1.1Jun 2, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Feb 16, 2022Group: Database references / Structure summary / Category: audit_author / database_2
Item: _audit_author.name / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: DNA-directed RNA polymerase subunit
B: DNA-directed RNA polymerase subunit beta
C: DNA-directed RNA polymerase II subunit RPB3
E: DNA-directed RNA polymerase II subunit E
F: DNA-directed RNA polymerase II subunit F
H: DNA-directed RNA polymerases I, II, and III subunit RPABC3
I: DNA-directed RNA polymerase II subunit RPB9
J: Uncharacterized Protein
K: RNA_pol_L_2 domain-containing protein
L: RNA polymerase II subunit K
M: Putative RNA polymerase II subunit B1 CTD phosphatase RPAP2
N: Unknown
hetero molecules


Theoretical massNumber of molelcules
Total (without water)553,87618
Polymers553,48312
Non-polymers3926
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area47930 Å2
ΔGint-262 kcal/mol
Surface area122980 Å2
MethodPISA

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Components

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DNA-directed RNA polymerase ... , 6 types, 6 molecules ABCEFI

#1: Protein DNA-directed RNA polymerase subunit


Mass: 217450.078 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus / References: UniProt: I3LJR4, DNA-directed RNA polymerase
#2: Protein DNA-directed RNA polymerase subunit beta


Mass: 134041.422 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus
References: UniProt: A0A0B8RVL1, DNA-directed RNA polymerase
#3: Protein DNA-directed RNA polymerase II subunit RPB3 / RNA polymerase II subunit C


Mass: 30997.557 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus / References: UniProt: I3LCH3
#4: Protein DNA-directed RNA polymerase II subunit E / RPB5 homolog


Mass: 24644.318 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus / References: UniProt: I3LSI7
#5: Protein DNA-directed RNA polymerase II subunit F / DNA-directed RNA polymerases I / II / and III subunit RPABC2 / RPB6 homolog


Mass: 14477.001 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus / References: UniProt: F1SKN8
#7: Protein DNA-directed RNA polymerase II subunit RPB9 / RNA polymerase II subunit B9 / DNA-directed RNA polymerase II subunit I / RNA polymerase II 14.5 ...RNA polymerase II subunit B9 / DNA-directed RNA polymerase II subunit I / RNA polymerase II 14.5 kDa subunit / RPB14.5


Mass: 14541.221 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus / References: UniProt: P60899

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Protein , 5 types, 5 molecules HJKLM

#6: Protein DNA-directed RNA polymerases I, II, and III subunit RPABC3


Mass: 17162.273 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus / References: UniProt: A0A4X1ULF2
#8: Protein Uncharacterized Protein


Mass: 7655.123 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus
#9: Protein RNA_pol_L_2 domain-containing protein


Mass: 13310.284 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus / References: UniProt: A0A4X1UK25
#10: Protein RNA polymerase II subunit K


Mass: 7018.244 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Sus scrofa domesticus (domestic pig) / Tissue: Thymus / References: UniProt: I3LN51
#11: Protein Putative RNA polymerase II subunit B1 CTD phosphatase RPAP2 / RNA polymerase II-associated protein 2


Mass: 69614.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: RPAP2, C1orf82 / Production host: Trichoplusia ni (cabbage looper)
References: UniProt: Q8IXW5, protein-serine/threonine phosphatase

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Protein/peptide / Non-polymers , 2 types, 7 molecules N

#12: Protein/peptide Unknown


Mass: 2571.161 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Trichoplusia ni (cabbage looper)
#13: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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
IDNameTypeEntity IDParent-IDSource
1RNA polymerase II -RPAP2 complexCOMPLEX#1-#120MULTIPLE SOURCES
2DNA-directed RNA polymerase IICOMPLEX#1-#101NATURAL
3Putative RNA polymerase II subunit B1 CTD phosphatase RPAP2COMPLEX#11-#121RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Sus scrofa domesticus (domestic pig)9825
23Homo sapiens (human)9606
Source (recombinant)Organism: Trichoplusia ni (cabbage looper)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R2/2
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER
Image recordingAverage exposure time: 2.21 sec. / Electron dose: 42.4 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

SoftwareName: PHENIX / Version: dev_3942: / Classification: refinement
EM software
IDNameVersionCategory
1Warpparticle selection
4WarpCTF correction
7UCSF Chimera1.13model fitting
9PHENIX1.14model refinement
11RELION3.0.5final Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1556776
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 364771 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5FLM

5flm


Pdb chain-ID: A
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00225553
ELECTRON MICROSCOPYf_angle_d0.54634530
ELECTRON MICROSCOPYf_dihedral_angle_d10.0773422
ELECTRON MICROSCOPYf_chiral_restr0.0443883
ELECTRON MICROSCOPYf_plane_restr0.0034462

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