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- PDB-3j1o: Cryo-EM map of a yeast minimal preinitiation complex interacting ... -

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Basic information

Entry
Database: PDB / ID: 3j1o
TitleCryo-EM map of a yeast minimal preinitiation complex interacting with the Mediator Head module
Components(Mediator of RNA polymerase II transcription subunit ...) x 7
KeywordsTRANSCRIPTION / RNA polymerase II / mediator head module / preinitiation complex
Function / homology
Function and homology information


RNA polymerase II complex recruiting activity / TFIIH-class transcription factor complex binding / core mediator complex / negative regulation of ribosomal protein gene transcription by RNA polymerase II / transcription open complex formation at RNA polymerase II promoter / mediator complex / termination of RNA polymerase II transcription / TFIID-class transcription factor complex binding / positive regulation of transcription initiation by RNA polymerase II / RNA polymerase II core promoter sequence-specific DNA binding ...RNA polymerase II complex recruiting activity / TFIIH-class transcription factor complex binding / core mediator complex / negative regulation of ribosomal protein gene transcription by RNA polymerase II / transcription open complex formation at RNA polymerase II promoter / mediator complex / termination of RNA polymerase II transcription / TFIID-class transcription factor complex binding / positive regulation of transcription initiation by RNA polymerase II / RNA polymerase II core promoter sequence-specific DNA binding / RNA polymerase II preinitiation complex assembly / TBP-class protein binding / transcription initiation at RNA polymerase II promoter / positive regulation of transcription elongation by RNA polymerase II / transcription coregulator activity / transcription corepressor activity / protein-macromolecule adaptor activity / DNA-binding transcription factor binding / transcription coactivator activity / RNA polymerase II cis-regulatory region sequence-specific DNA binding / protein domain specific binding / regulation of transcription by RNA polymerase II / negative regulation of transcription by RNA polymerase II / positive regulation of transcription by RNA polymerase II / nucleus
Similarity search - Function
Mediator of RNA polymerase II transcription subunit 22, Saccharomycetes / Mediator of RNA polymerase II transcription subunit 20 / Mediator complex, subunit Med6, fungi / Mediator complex, subunit Med20 / TATA-binding related factor (TRF) of subunit 20 of Mediator complex / Mediator complex, subunit Med6 / Mediator complex, subunit Med17 / Mediator complex, subunit Med6 superfamily / MED6 mediator sub complex component / Subunit 17 of Mediator complex ...Mediator of RNA polymerase II transcription subunit 22, Saccharomycetes / Mediator of RNA polymerase II transcription subunit 20 / Mediator complex, subunit Med6, fungi / Mediator complex, subunit Med20 / TATA-binding related factor (TRF) of subunit 20 of Mediator complex / Mediator complex, subunit Med6 / Mediator complex, subunit Med17 / Mediator complex, subunit Med6 superfamily / MED6 mediator sub complex component / Subunit 17 of Mediator complex / Mediator of RNA polymerase II transcription subunit 22 / Mediator complex, subunit Med8, fungi/metazoa / Mediator complex, subunit Med11 / Surfeit locus protein 5 subunit 22 of Mediator complex / Mediator of RNA polymerase II transcription complex subunit 8 / Mediator complex protein / Mediator complex, subunit Med18 / Med18 protein
Similarity search - Domain/homology
: / Mediator of RNA polymerase II transcription subunit 17 / Mediator of RNA polymerase II transcription subunit 22 / Mediator of RNA polymerase II transcription subunit 18 / Mediator of RNA polymerase II transcription subunit 20 / Mediator of RNA polymerase II transcription subunit 8 / Mediator of RNA polymerase II transcription subunit 6 / Mediator of RNA polymerase II transcription subunit 11
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 16 Å
AuthorsAsturias, F.J. / Imasaki, T.
CitationJournal: Structure / Year: 2012
Title: Interaction of the mediator head module with RNA polymerase II.
Authors: Gang Cai / Yuriy L Chaban / Tsuyoshi Imasaki / Julio A Kovacs / Guillermo Calero / Pawel A Penczek / Yuichiro Takagi / Francisco J Asturias /
Abstract: Mediator, a large (21 polypeptides, MW ∼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the ...Mediator, a large (21 polypeptides, MW ∼1 MDa) complex conserved throughout eukaryotes, plays an essential role in control of gene expression by conveying regulatory signals that influence the activity of the preinitiation complex. However, the precise mode of interaction between Mediator and RNA polymerase II (RNAPII), and the mechanism of regulation by Mediator remain elusive. We used cryo-electron microscopy and reconstituted in vitro transcription assays to characterize a transcriptionally-active complex including the Mediator Head module and components of a minimum preinitiation complex (RNAPII, TFIIF, TFIIB, TBP, and promoter DNA). Our results reveal how the Head interacts with RNAPII, affecting its conformation and function.
History
DepositionMar 29, 2012Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2013Provider: repository / Type: Initial release
Revision 1.1Aug 19, 2015Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id
Revision 1.3Feb 21, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_ref_seq_dif
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_ref_seq_dif.details

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Structure visualization

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Assembly

Deposited unit
H: Mediator of RNA polymerase II transcription subunit 11
I: Mediator of RNA polymerase II transcription subunit 17
J: Mediator of RNA polymerase II transcription subunit 8
K: Mediator of RNA polymerase II transcription subunit 22
L: Mediator of RNA polymerase II transcription subunit 18
M: Mediator of RNA polymerase II transcription subunit 20
N: Mediator of RNA polymerase II transcription subunit 6


Theoretical massNumber of molelcules
Total (without water)165,6947
Polymers165,6947
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Mediator of RNA polymerase II transcription subunit ... , 7 types, 7 molecules HIJKLMN

#1: Protein Mediator of RNA polymerase II transcription subunit 11 / Mediator complex subunit 11


Mass: 15100.063 Da / Num. of mol.: 1 / Mutation: T16G, M17S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MED11, YMR112C, YM9718.11C / Plasmid: pBakPAC / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: A0A0D3YMY9, UniProt: Q99278*PLUS
#2: Protein Mediator of RNA polymerase II transcription subunit 17 / Mediator complex subunit 17 / Suppressor of RNA polymerase B 4


Mass: 54893.527 Da / Num. of mol.: 1 / Fragment: SEE REMARK 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SRB4, MED17, YER022W / Plasmid: pBakPAC / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P32569
#3: Protein Mediator of RNA polymerase II transcription subunit 8 / Mediator complex subunit 8


Mass: 25297.234 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MED8, YBR193C, YBR1403 / Plasmid: pBakPAC / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P38304
#4: Protein Mediator of RNA polymerase II transcription subunit 22 / Mediator complex subunit 22 / Suppressor of RNA polymerase B 6


Mass: 13875.731 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SRB6, MED22, YBR253W, YBR1721 / Plasmid: pBakPAC / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P32570
#5: Protein Mediator of RNA polymerase II transcription subunit 18 / Mediator complex subunit 18 / Suppressor of RNA polymerase B 5


Mass: 30659.801 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SRB5, MED18, YGR104C / Plasmid: pBakPAC / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P32585
#6: Protein Mediator of RNA polymerase II transcription subunit 20 / Hyper-recombination suppressor protein 2 / Mediator complex subunit 20 / Suppressor of RNA polymerase B 2


Mass: 22918.068 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SRB2, HRS2, MED20, YHR041C / Plasmid: pBakPAC / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P34162
#7: Protein/peptide Mediator of RNA polymerase II transcription subunit 6 / Mediator complex subunit 6


Mass: 2949.360 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: MED6, MTR32, YHR058C / Plasmid: pBakPAC / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P38782

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Details

Sequence detailsTHE ARRANGEMENT OF RESIDUES 617-668 OF CHAIN I IS UNCLEAR. THESE RESIDUES HAVE BEEN DEFINED AS UNK ...THE ARRANGEMENT OF RESIDUES 617-668 OF CHAIN I IS UNCLEAR. THESE RESIDUES HAVE BEEN DEFINED AS UNK (UNKNOWN RESIDUE) IN THE COORDINATES AND NUMBERED 1094-1101, 1028-1038, 1316-1323, 1619-1627, AND 1719-1727 IN ANALOGY TO PDB ENTRY 3RJ1.

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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
IDNameTypeDetailsParent-ID
1Cryo-EM map of a complex including RNA polymerase II, Mediator Head module, TFIIF, TFIIB, TBP, and promoter DNACOMPLEXAlthough biochemical and functional evidence indicates the presence of all 6 original components (RNA polymerase II, Mediator Head, TFIIF, TFIIB, TBP, DNA), only 3 components (RNA polymerase II, Mediator Head, TFIIF) can be clearly identified in the cryo-EM map.0
2mediator head module1
3Head module1
Molecular weightValue: 0.9 MDa / Experimental value: NO
Buffer solutionName: 25 mM KCl, 25 mM Tris-HCl, 10 mM DTT / pH: 8 / Details: 25 mM KCl, 25 mM Tris-HCl, 10 mM DTT
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 25mM KCl, 25mM Tris-HCl, 10mM DTT
Specimen supportDetails: 400 mesh Cu/Rh grids, coated with a perforated carbon film and glow discharged in the presence of amylamine
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temp: 77 K / Humidity: 95 %
Details: Blot for approximately 2 seconds before plunging into liquid ethane.
Method: Blot for ~2 sec before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Jul 30, 2008
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 50000 X / Nominal defocus max: 3800 nm / Nominal defocus min: 800 nm / Cs: 2 mm
Astigmatism: Objective lens astigmatisma was corrected at 125,000X magnification
Camera length: 0 mm
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Temperature: 115 K / Temperature (max): 120 K / Temperature (min): 77 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 10 e/Å2 / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k)
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategoryDetails
1OTHERmodel fittingMonte Carlo Docking Analysis
2SPARX3D reconstruction
3SPIDER3D reconstruction
CTF correctionDetails: Each CCD frame
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: projection matching / Resolution: 16 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 51000 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1RIGID BODY FITREALREFINEMENT PROTOCOL--Rigid body fitting of individual RNA polymerase II structural modules DETAILS--Fitted individual structural modules previously identified by X-ray crystallographic studies of RNA polymerase II under different crystallization conditions
2RIGID BODY FITREALREFINEMENT PROTOCOL--Rigid body fitting of individual Mediator Head structural modules DETAILS--Fitted individual structural modules previously identified by EM and X-ray crystallographic studies of the Head module
Atomic model building

3D fitting-ID: 1 / Accession code: 3RJ1 / Initial refinement model-ID: 1 / PDB-ID: 3RJ1

/ Source name: PDB / Type: experimental model

IDPdb chain-ID
1A
2B
3C
4D
5E
6F
7G
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms5560 0 0 0 5560

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