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- EMDB-16365: RNA polymerase II core pre-initiation complex focused map -

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Basic information

Entry
Database: EMDB / ID: EMD-16365
TitleRNA polymerase II core pre-initiation complex focused map
Map data
Sample
  • Complex: RNA polymerase II core pre-initiation complex
    • Complex: General transcription factor IIH
    • Complex: Mammalian RNA polymerase II
    • Complex: General transcription factor IIB
    • Complex: TATA-box binding protein
    • Complex: General transcription factor IIF
    • Complex: General transcription factor IIA
    • Complex: General transcription factor IIE
KeywordsMammalian PIC / +1 nucleosome / transcription initiation / TRANSCRIPTION
Biological speciesHomo sapiens (human) / Sus scrofa (pig)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsAbril-Garrido J / Dienemann C / Grabbe F / Velychko T / Lidschreiber M / Wang H / Cramer P
Funding support Germany, European Union, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG) Germany
European Research Council (ERC)European Union
CitationJournal: Mol Cell / Year: 2023
Title: Structural basis of transcription reduction by a promoter-proximal +1 nucleosome.
Authors: Julio Abril-Garrido / Christian Dienemann / Frauke Grabbe / Taras Velychko / Michael Lidschreiber / Haibo Wang / Patrick Cramer /
Abstract: At active human genes, the +1 nucleosome is located downstream of the RNA polymerase II (RNA Pol II) pre-initiation complex (PIC). However, at inactive genes, the +1 nucleosome is found further ...At active human genes, the +1 nucleosome is located downstream of the RNA polymerase II (RNA Pol II) pre-initiation complex (PIC). However, at inactive genes, the +1 nucleosome is found further upstream, at a promoter-proximal location. Here, we establish a model system to show that a promoter-proximal +1 nucleosome can reduce RNA synthesis in vivo and in vitro, and we analyze its structural basis. We find that the PIC assembles normally when the edge of the +1 nucleosome is located 18 base pairs (bp) downstream of the transcription start site (TSS). However, when the nucleosome edge is located further upstream, only 10 bp downstream of the TSS, the PIC adopts an inhibited state. The transcription factor IIH (TFIIH) shows a closed conformation and its subunit XPB contacts DNA with only one of its two ATPase lobes, inconsistent with DNA opening. These results provide a mechanism for nucleosome-dependent regulation of transcription initiation.
History
DepositionDec 16, 2022-
Header (metadata) releaseMay 3, 2023-
Map releaseMay 3, 2023-
UpdateJun 14, 2023-
Current statusJun 14, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16365.png

Downloads & links

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Map

FileDownload / File: emd_16365.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 300 pix.
= 315. Å		1.05 Å/pix.
x 300 pix.
= 315. Å		1.05 Å/pix.
x 300 pix.
= 315. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.008
Minimum - Maximum-0.018110348 - 0.048713893
Average (Standard dev.)-0.000049670238 (±0.0022804025)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 315.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16365_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_16365_additional_1.map
Projections & Slices
AxesZYX

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Histogram

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Half map: #1

Fileemd_16365_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_16365_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : RNA polymerase II core pre-initiation complex

EntireName: RNA polymerase II core pre-initiation complex
Components
  • Complex: RNA polymerase II core pre-initiation complex
    • Complex: General transcription factor IIH
    • Complex: Mammalian RNA polymerase II
    • Complex: General transcription factor IIB
    • Complex: TATA-box binding protein
    • Complex: General transcription factor IIF
    • Complex: General transcription factor IIA
    • Complex: General transcription factor IIE

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Supramolecule #1: RNA polymerase II core pre-initiation complex

SupramoleculeName: RNA polymerase II core pre-initiation complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#24, #26-#30
Molecular weightTheoretical: 800 KDa

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Supramolecule #2: General transcription factor IIH

SupramoleculeName: General transcription factor IIH / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#10
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Mammalian RNA polymerase II

SupramoleculeName: Mammalian RNA polymerase II / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #11-#22
Source (natural)Organism: Sus scrofa (pig)

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Supramolecule #4: General transcription factor IIB

SupramoleculeName: General transcription factor IIB / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #23
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #5: TATA-box binding protein

SupramoleculeName: TATA-box binding protein / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #24
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #6: General transcription factor IIF

SupramoleculeName: General transcription factor IIF / type: complex / ID: 6 / Parent: 1 / Macromolecule list: #25-#26
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #7: General transcription factor IIA

SupramoleculeName: General transcription factor IIA / type: complex / ID: 7 / Parent: 1 / Macromolecule list: #27-#28
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #8: General transcription factor IIE

SupramoleculeName: General transcription factor IIE / type: complex / ID: 8 / Parent: 1 / Macromolecule list: #29-#30
Source (natural)Organism: Homo sapiens (human)

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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

BufferpH: 7.5
GridModel: Quantifoil R3.5/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 3
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum SE / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 41517 / Average exposure time: 3.0 sec. / Average electron dose: 41.58 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4667603
Startup modelType of model: OTHER
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 188832
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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