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- EMDB-2784: Architecture of the RNA polymerase II-Mediator core transcription... -

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Basic information

Entry
Database: EMDB / ID: EMD-2784
TitleArchitecture of the RNA polymerase II-Mediator core transcription initiation complex
Map dataPol II-DNA/RNA
Sample
  • Sample: Pol II-DNA/RNA
  • Protein or peptide: x 12 types
  • DNA: x 2 types
  • RNA: x 1 types
Keywordstranscription / transcription initiation / RNA polymerase II / General Transcription Factors
Function / homology
Function and homology information


RPB4-RPB7 complex / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / RNA polymerase II transcribes snRNA genes / termination of RNA polymerase II transcription ...RPB4-RPB7 complex / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / RNA Polymerase I Transcription Initiation / Processing of Capped Intron-Containing Pre-mRNA / RNA Polymerase III Transcription Initiation From Type 2 Promoter / RNA Pol II CTD phosphorylation and interaction with CE / Formation of the Early Elongation Complex / mRNA Capping / RNA polymerase II transcribes snRNA genes / termination of RNA polymerase II transcription / TP53 Regulates Transcription of DNA Repair Genes / RNA Polymerase II Promoter Escape / RNA Polymerase II Transcription Pre-Initiation And Promoter Opening / RNA Polymerase II Transcription Initiation / RNA Polymerase II Transcription Initiation And Promoter Clearance / termination of RNA polymerase III transcription / RNA Polymerase II Pre-transcription Events / RNA-templated transcription / positive regulation of nuclear-transcribed mRNA poly(A) tail shortening / Formation of TC-NER Pre-Incision Complex / transcription initiation at RNA polymerase III promoter / RNA Polymerase I Promoter Escape / termination of RNA polymerase I transcription / nucleolar large rRNA transcription by RNA polymerase I / Gap-filling DNA repair synthesis and ligation in TC-NER / transcription initiation at RNA polymerase I promoter / Estrogen-dependent gene expression / maintenance of transcriptional fidelity during transcription elongation by RNA polymerase II / nuclear-transcribed mRNA catabolic process / positive regulation of translational initiation / transcription by RNA polymerase III / Dual incision in TC-NER / translesion synthesis / RNA polymerase I complex / transcription elongation by RNA polymerase I / RNA polymerase III complex / RNA polymerase II, core complex / tRNA transcription by RNA polymerase III / transcription by RNA polymerase I / translation initiation factor binding / transcription-coupled nucleotide-excision repair / transcription initiation at RNA polymerase II promoter / transcription elongation by RNA polymerase II / P-body / DNA-templated transcription initiation / ribonucleoside binding / mRNA processing / DNA-directed RNA polymerase / cytoplasmic stress granule / DNA-directed RNA polymerase activity / peroxisome / ribosome biogenesis / single-stranded DNA binding / nucleic acid binding / transcription by RNA polymerase II / single-stranded RNA binding / protein dimerization activity / nucleotide binding / mRNA binding / nucleolus / mitochondrion / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytoplasm
Similarity search - Function
DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb1 C-terminal repeat / RNA polymerase II, heptapeptide repeat, eukaryotic / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / RNA polymerase Rpb1, domain 7 / RNA polymerase Rpb1, domain 7 superfamily ...DNA-directed RNA polymerase II subunit Rpb4-like / RNA polymerase Rpb1 C-terminal repeat / RNA polymerase II, heptapeptide repeat, eukaryotic / Eukaryotic RNA polymerase II heptapeptide repeat. / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb1, domain 6 / RNA polymerase Rpb4/RPC9, core / DNA-directed RNA-polymerase II subunit / 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 / Rpb4/RPC9 superfamily / RNA polymerase subunit Rpb4/RPC9 / RNA polymerase Rpb4 / Zinc finger TFIIS-type signature. / HRDC-like superfamily / RNA polymerase Rpb7-like , N-terminal / RNA polymerase Rpb7-like, N-terminal domain superfamily / RNA polymerase subunit Rpb7-like / SHS2 domain found in N terminus of Rpb7p/Rpc25p/MJ0397 / 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 subunit RPABC5/Rpb10 / RNA polymerases, subunit N, zinc binding site / RNA polymerase subunit RPB10 / RNA polymerases N / 8 kDa subunit / RNA polymerases N / 8 Kd subunits signature. / 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. / RNA polymerase subunit RPABC4/transcription elongation factor Spt4 / Zinc finger, TFIIS-type / Transcription factor S-II (TFIIS) / Zinc finger TFIIS-type profile. / C2C2 Zinc finger / DNA-directed RNA polymerase, RBP11-like dimerisation domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerase, subunit H/Rpb5 C-terminal / DNA-directed RNA polymerase subunit Rpo5/Rpb5 / 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. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 3 superfamily / RPB6/omega subunit-like superfamily / 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 Rpb1, clamp domain superfamily / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / 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 / DNA-directed RNA polymerase, insert domain superfamily
Similarity search - Domain/homology
DNA-directed RNA polymerase II subunit RPB1 / DNA-directed RNA polymerase II subunit RPB2 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase II subunit RPB4 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase II subunit RPB9 / DNA-directed RNA polymerase II subunit RPB7 ...DNA-directed RNA polymerase II subunit RPB1 / DNA-directed RNA polymerase II subunit RPB2 / DNA-directed RNA polymerase II subunit RPB3 / DNA-directed RNA polymerase II subunit RPB4 / DNA-directed RNA polymerases I, II, and III subunit RPABC1 / DNA-directed RNA polymerases I, II, and III subunit RPABC2 / DNA-directed RNA polymerases I, II, and III subunit RPABC3 / DNA-directed RNA polymerases I, II, and III subunit RPABC5 / DNA-directed RNA polymerase II subunit RPB9 / DNA-directed RNA polymerase II subunit RPB7 / DNA-directed RNA polymerase II subunit RPB11 / DNA-directed RNA polymerases I, II, and III subunit RPABC4
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsPlaschka C / Lariviere L / Wenzeck L / Hemann M / Tegunov D / Petrotchenko EV / Borchers CH / Baumeister W / Herzog F / Villa E / Cramer P
CitationJournal: Nature / Year: 2015
Title: Architecture of the RNA polymerase II-Mediator core initiation complex.
Authors: C Plaschka / L Larivière / L Wenzeck / M Seizl / M Hemann / D Tegunov / E V Petrotchenko / C H Borchers / W Baumeister / F Herzog / E Villa / P Cramer /
Abstract: The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all ...The conserved co-activator complex Mediator enables regulated transcription initiation by RNA polymerase (Pol) II. Here we reconstitute an active 15-subunit core Mediator (cMed) comprising all essential Mediator subunits from Saccharomyces cerevisiae. The cryo-electron microscopic structure of cMed bound to a core initiation complex was determined at 9.7 Å resolution. cMed binds Pol II around the Rpb4-Rpb7 stalk near the carboxy-terminal domain (CTD). The Mediator head module binds the Pol II dock and the TFIIB ribbon and stabilizes the initiation complex. The Mediator middle module extends to the Pol II foot with a 'plank' that may influence polymerase conformation. The Mediator subunit Med14 forms a 'beam' between the head and middle modules and connects to the tail module that is predicted to bind transcription activators located on upstream DNA. The Mediator 'arm' and 'hook' domains contribute to a 'cradle' that may position the CTD and TFIIH kinase to stimulate Pol II phosphorylation.
History
DepositionSep 21, 2014-
Header (metadata) releaseOct 29, 2014-
Map releaseFeb 4, 2015-
UpdateMar 4, 2015-
Current statusMar 4, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4v1m
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2784.png

Downloads & links

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Map

FileDownload / File: emd_2784.map.gz / Format: CCP4 / Size: 81.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPol II-DNA/RNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 280 pix.
= 378. Å		1.35 Å/pix.
x 280 pix.
= 378. Å		1.35 Å/pix.
x 280 pix.
= 378. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.04966678 - 0.10112445
Average (Standard dev.)0.00006485 (±0.00348376)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 378.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-32-96
NX/NY/NZ8165193
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0500.1010.000

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Supplemental data

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Sample components

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Entire : Pol II-DNA/RNA

EntireName: Pol II-DNA/RNA
Components
  • Sample: Pol II-DNA/RNA
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB4
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 1
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 2
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 3
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB9
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 5
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB11
  • Protein or peptide: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 4
  • DNA: template DNA
  • DNA: nontemplate DNA
  • RNA: RNA

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Supramolecule #1000: Pol II-DNA/RNA

SupramoleculeName: Pol II-DNA/RNA / type: sample / ID: 1000 / Number unique components: 15
Molecular weightTheoretical: 570 KDa

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Macromolecule #1: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB1 / type: protein_or_peptide / ID: 1 / Name.synonym: RPO21 RPB1 RPB220 SUA8 YDL140C D2150 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 192 KDa / Theoretical: 192 KDa
SequenceUniProtKB: DNA-directed RNA polymerase II subunit RPB1

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Macromolecule #2: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB2 / type: protein_or_peptide / ID: 2 / Name.synonym: RPB2 RPB150 RPO22 YOR151C / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 139 KDa / Theoretical: 139 KDa
SequenceUniProtKB: DNA-directed RNA polymerase II subunit RPB2

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Macromolecule #3: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB3 / type: protein_or_peptide / ID: 3 / Name.synonym: RPB3 YIL021W / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 35 KDa / Theoretical: 35 KDa
SequenceUniProtKB: DNA-directed RNA polymerase II subunit RPB3

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Macromolecule #4: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB4

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB4 / type: protein_or_peptide / ID: 4 / Name.synonym: RPB4 YJL140W J0654 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 25 KDa / Theoretical: 25 KDa
SequenceUniProtKB: DNA-directed RNA polymerase II subunit RPB4

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Macromolecule #5: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 1

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 1
type: protein_or_peptide / ID: 5 / Name.synonym: RPB5 RPA7 RPC9 YBR154C YBR1204 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 25 KDa / Theoretical: 25 KDa
SequenceUniProtKB: DNA-directed RNA polymerases I, II, and III subunit RPABC1

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Macromolecule #6: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 2

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 2
type: protein_or_peptide / ID: 6 / Name.synonym: RPO26 RPB6 YPR187W P9677.8 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 18 KDa / Theoretical: 18 KDa
SequenceUniProtKB: DNA-directed RNA polymerases I, II, and III subunit RPABC2

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Macromolecule #7: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB7 / type: protein_or_peptide / ID: 7 / Name.synonym: RPB7 YDR404C D9509.22 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 19 KDa / Theoretical: 19 KDa
SequenceUniProtKB: DNA-directed RNA polymerase II subunit RPB7

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Macromolecule #8: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 3

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 3
type: protein_or_peptide / ID: 8 / Name.synonym: RPB8 YOR224C YOR50-14 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 17 KDa / Theoretical: 17 KDa
SequenceUniProtKB: DNA-directed RNA polymerases I, II, and III subunit RPABC3

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Macromolecule #9: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB9

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB9 / type: protein_or_peptide / ID: 9 / Name.synonym: RPB9 YGL070C / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 14 KDa / Theoretical: 14 KDa
SequenceUniProtKB: DNA-directed RNA polymerase II subunit RPB9

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Macromolecule #10: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 5

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 5
type: protein_or_peptide / ID: 10 / Name.synonym: RPB10 YOR210W / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 8 KDa / Theoretical: 8 KDa
SequenceUniProtKB: DNA-directed RNA polymerases I, II, and III subunit RPABC5

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Macromolecule #11: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB11

MacromoleculeName: DNA-DIRECTED RNA POLYMERASE II SUBUNIT RPB11 / type: protein_or_peptide / ID: 11 / Name.synonym: RPB11 YOL005C / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 13 KDa / Theoretical: 13 KDa
SequenceUniProtKB: DNA-directed RNA polymerase II subunit RPB11

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Macromolecule #12: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 4

MacromoleculeName: DNA-DIRECTED RNA POLYMERASES I, II, AND III SUBUNIT RPABC 4
type: protein_or_peptide / ID: 12 / Name.synonym: RPC10 RPB12 YHR143W-A YHR143BW / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BJ5464 Rpb3 His-Bio / synonym: Baker's yeast / Organelle: Nucleus
Molecular weightExperimental: 8 KDa / Theoretical: 8 KDa
SequenceUniProtKB: DNA-directed RNA polymerases I, II, and III subunit RPABC4

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Macromolecule #13: template DNA

MacromoleculeName: template DNA / type: dna / ID: 13 / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
SequenceString:
CGAGAACAGT AGCACGCTGT GTATATAATA GTGTGTTGTA CATAGCGGAG GTCGGTGGGG CACAACTGCG CT

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Macromolecule #14: nontemplate DNA

MacromoleculeName: nontemplate DNA / type: dna / ID: 14 / Classification: DNA / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
SequenceString:
AGCGCAGTTG TGCTATGATA TTTTTATGTA TGTACAACAC ACTATTATAT ACACAGCGTG CTACTGTTCT CG

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Macromolecule #15: RNA

MacromoleculeName: RNA / type: rna / ID: 15 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)
SequenceString:
AUAUCA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.15 mg/mL
BufferpH: 7.5
Details: 25 mM HEPES-KOH pH 7.5, 180 mM Potassium acetate, 5 % Glycerol, 5 mM DTT
GridDetails: Lacey carbon copper grids (Quantifoil)
VitrificationCryogen name: ETHANE / Instrument: OTHER
Method: Grids were glow-discharged for 20 s before deposition of 4 microliters sample and incubated for 30 s. Grids were washed twice with 4 microliters distilled water, blotted, and then vitrified.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 2972 / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37169 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 37000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsParticles were picked using EMAN2 and were processed using RELION 1.2.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: OTHER / Software - Name: RELION, 1.2
Details: The density was filtered according to local resolution and further a temperature factor of minus 240 A2 was applied.
Number images used: 14777

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Atomic model buiding 1

Initial modelPDB ID:

4a3d


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C / Chain - #3 - Chain ID: E / Chain - #4 - Chain ID: F / Chain - #5 - Chain ID: H / Chain - #6 - Chain ID: I / Chain - #7 - Chain ID: J / Chain - #8 - Chain ID: K / Chain - #9 - Chain ID: L
SoftwareName: SITUS
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4v1m:
Architecture of the RNA polymerase II-Mediator core transcription initiation complex

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