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- EMDB-3306: Structure of human TFIID-IIA bound to core promoter DNA (locally-... -

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Basic information

Entry
Database: EMDB / ID: EMD-3306
TitleStructure of human TFIID-IIA bound to core promoter DNA (locally-refined lobe C)
Map dataLocally-refined lobe C of human TFIID bound to super core promoter DNA
Sample
  • Sample: Lobe C of human TFIID-TFIIA complex bound to the downstream region of super core promoter DNA
  • Protein or peptide: General transcription factor IID
  • Protein or peptide: General transcription factor IIA
  • DNA: Super core promoter
KeywordsTFIID / TFIIA / transcription / RNA polymerase II / general transcription factors / preinitiation complex / core promoter / DNA binding
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.2 Å
AuthorsLouder RK / He Y / Lopez-Blanco JR / Fang J / Chacon P / Nogales E
CitationJournal: Nature / Year: 2016
Title: Structure of promoter-bound TFIID and model of human pre-initiation complex assembly.
Authors: Robert K Louder / Yuan He / José Ramón López-Blanco / Jie Fang / Pablo Chacón / Eva Nogales /
Abstract: The general transcription factor IID (TFIID) plays a central role in the initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-initiation complex (PIC) assembly at the ...The general transcription factor IID (TFIID) plays a central role in the initiation of RNA polymerase II (Pol II)-dependent transcription by nucleating pre-initiation complex (PIC) assembly at the core promoter. TFIID comprises the TATA-binding protein (TBP) and 13 TBP-associated factors (TAF1-13), which specifically interact with a variety of core promoter DNA sequences. Here we present the structure of human TFIID in complex with TFIIA and core promoter DNA, determined by single-particle cryo-electron microscopy at sub-nanometre resolution. All core promoter elements are contacted by subunits of TFIID, with TAF1 and TAF2 mediating major interactions with the downstream promoter. TFIIA bridges the TBP-TATA complex with lobe B of TFIID. We also present the cryo-electron microscopy reconstruction of a fully assembled human TAF-less PIC. Superposition of common elements between the two structures provides novel insights into the general role of TFIID in promoter recognition, PIC assembly, and transcription initiation.
History
DepositionJan 27, 2016-
Header (metadata) releaseFeb 3, 2016-
Map releaseMar 30, 2016-
UpdateApr 13, 2016-
Current statusApr 13, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3306.png

Downloads & links

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Map

FileDownload / File: emd_3306.map.gz / Format: CCP4 / Size: 210.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally-refined lobe C of human TFIID bound to super core promoter DNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.32 Å/pix.
x 384 pix.
= 506.88 Å		1.32 Å/pix.
x 384 pix.
= 506.88 Å		1.32 Å/pix.
x 384 pix.
= 506.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.32 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.03581309 - 0.07569469
Average (Standard dev.)0.00005752 (±0.0011652)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 506.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z506.880506.880506.880
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-64
NX/NY/NZ6161129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0360.0760.000

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Supplemental data

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Sample components

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Entire : Lobe C of human TFIID-TFIIA complex bound to the downstream regio...

EntireName: Lobe C of human TFIID-TFIIA complex bound to the downstream region of super core promoter DNA
Components
  • Sample: Lobe C of human TFIID-TFIIA complex bound to the downstream region of super core promoter DNA
  • Protein or peptide: General transcription factor IID
  • Protein or peptide: General transcription factor IIA
  • DNA: Super core promoter

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Supramolecule #1000: Lobe C of human TFIID-TFIIA complex bound to the downstream regio...

SupramoleculeName: Lobe C of human TFIID-TFIIA complex bound to the downstream region of super core promoter DNA
type: sample / ID: 1000
Details: This reconstruction was obtained from a sample that contained the full TFIID, TFIIA, and super core promoter DNA, but TFIIA and parts of TFIID (lobes A and B) and promoter DNA (-40 to -5) ...Details: This reconstruction was obtained from a sample that contained the full TFIID, TFIIA, and super core promoter DNA, but TFIIA and parts of TFIID (lobes A and B) and promoter DNA (-40 to -5) have been masked out in this reconstruction.
Number unique components: 3
Molecular weightTheoretical: 1.34 MDa

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Macromolecule #1: General transcription factor IID

MacromoleculeName: General transcription factor IID / type: protein_or_peptide / ID: 1 / Name.synonym: TFIID / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa / synonym: Human / Organelle: Nucleus / Location in cell: Nuclear extract
Molecular weightTheoretical: 1.26 MDa

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Macromolecule #2: General transcription factor IIA

MacromoleculeName: General transcription factor IIA / type: protein_or_peptide / ID: 2 / Name.synonym: TFIIA / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightTheoretical: 27 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #3: Super core promoter

MacromoleculeName: Super core promoter / type: dna / ID: 3 / Name.synonym: SCP
Details: The super core promoter is a composite sequence combining promoter motifs from several strong promoters from humans and D. melanogaster.
Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 56 KDa
SequenceString:
GAAGGGCGCC TATAAAAGGG GGTGGGGGCG CGTTCGTCCT CAGTCGCGAT CGAACACTCG AGCCGAGCAG ACGTGCCTAC GGACCATGG

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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

Concentration0.05 mg/mL
BufferpH: 7.9
Details: 10 mM HEPES, 10 mM MgCl2, 50 mM KCl, 3% trehalose 1 mM DTT, 0.0125% NP-40
GridDetails: Amorphous continuous carbon over C-flat holey carbon support (4 um holes with 2 um spacing) on 400 mesh copper grid.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
Method: Incubate 4 ul of sample on grid for 10 minutes, then blot for 4 seconds with force 15.

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Electron microscopy

MicroscopeFEI TITAN
DetailsThe camera was operated in counting mode with a dose rate of 8 electrons/pixel per second, with a total exposure time of 10 seconds fractionated over 20 frames.
DateAug 11, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 1253
Details: Whole-micrograph drift correction was performed using MotionCorr before averaging the frames.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37879 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN

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Image processing

DetailsFinal rounds of 3D image classification and refinement were performed within a mask around the lobe C of TFIID and bound downstream promoter DNA.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.2 Å / Resolution method: OTHER / Software - Name: CTFFIND4, RELION, Bsoft
Details: After whole micrograph drift correction, per-particle beam-induced motion and radiation damage were corrected using the particle polishing procedure within RELION. Final map was filtered ...Details: After whole micrograph drift correction, per-particle beam-induced motion and radiation damage were corrected using the particle polishing procedure within RELION. Final map was filtered according to local resolution using Bsoft.
Number images used: 28448
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4rgw

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

4atg

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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