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- EMDB-2283: Human TFIID Binds to Core Promoter DNA in a Reorganized Structura... -

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Basic information

Entry
Database: EMDB / ID: EMD-2283
TitleHuman TFIID Binds to Core Promoter DNA in a Reorganized Structural State
Map data3D reconstruction of canonical conformation of human TFIID in the presence of TFIIA and super core promoter DNA at 32A
Sample
  • Sample: TFIID-TFIIA-SCP (canonical)
  • Protein or peptide: Transcription factor II-D
  • Protein or peptide: Transcription factor II-A
  • Protein or peptide: Transcription factor SCP
KeywordsTFIID / transcription / cryo-electron microscopy / TFIIA / core promoter / RNA polymerase II
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 32.0 Å
AuthorsCianfrocco MA / Kassavetis GA / Grob P / Fang J / Juven-Gershon T / Kadonaga JT / Nogales E
CitationJournal: Cell / Year: 2013
Title: Human TFIID binds to core promoter DNA in a reorganized structural state.
Authors: Michael A Cianfrocco / George A Kassavetis / Patricia Grob / Jie Fang / Tamar Juven-Gershon / James T Kadonaga / Eva Nogales /
Abstract: A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step ...A mechanistic description of metazoan transcription is essential for understanding the molecular processes that govern cellular decisions. To provide structural insights into the DNA recognition step of transcription initiation, we used single-particle electron microscopy (EM) to visualize human TFIID with promoter DNA. This analysis revealed that TFIID coexists in two predominant and distinct structural states that differ by a 100 Å translocation of TFIID's lobe A. The transition between these structural states is modulated by TFIIA, as the presence of TFIIA and promoter DNA facilitates the formation of a rearranged state of TFIID that enables promoter recognition and binding. DNA labeling and footprinting, together with cryo-EM studies, were used to map the locations of TATA, Initiator (Inr), motif ten element (MTE), and downstream core promoter element (DPE) promoter motifs within the TFIID-TFIIA-DNA structure. The existence of two structurally and functionally distinct forms of TFIID suggests that the different conformers may serve as specific targets for the action of regulatory factors.
History
DepositionJan 17, 2013-
Header (metadata) releaseJan 23, 2013-
Map releaseJan 23, 2013-
UpdateJan 30, 2013-
Current statusJan 30, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.128
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.128
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2283.png

Downloads & links

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Map

FileDownload / File: emd_2283.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of canonical conformation of human TFIID in the presence of TFIIA and super core promoter DNA at 32A
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.48 Å/pix.
x 100 pix.
= 548. Å		5.48 Å/pix.
x 100 pix.
= 548. Å		5.48 Å/pix.
x 100 pix.
= 548. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.48 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.128 / Movie #1: 0.128
Minimum - Maximum-0.22743234 - 0.74917156
Average (Standard dev.)-0.00323071 (±0.03499755)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 548.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.485.485.48
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z548.000548.000548.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-36-30-80
NX/NY/NZ7361161
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.2270.749-0.003

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Supplemental data

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Sample components

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Entire : TFIID-TFIIA-SCP (canonical)

EntireName: TFIID-TFIIA-SCP (canonical)
Components
  • Sample: TFIID-TFIIA-SCP (canonical)
  • Protein or peptide: Transcription factor II-D
  • Protein or peptide: Transcription factor II-A
  • Protein or peptide: Transcription factor SCP

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Supramolecule #1000: TFIID-TFIIA-SCP (canonical)

SupramoleculeName: TFIID-TFIIA-SCP (canonical) / type: sample / ID: 1000 / Number unique components: 3
Molecular weightExperimental: 1.4 MDa / Theoretical: 1.4 MDa

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Macromolecule #1: Transcription factor II-D

MacromoleculeName: Transcription factor II-D / type: protein_or_peptide / ID: 1 / Name.synonym: TFIID / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa / synonym: Human / Organelle: Nucleus
Molecular weightExperimental: 1.4 MDa / Theoretical: 1.4 MDa

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Macromolecule #2: Transcription factor II-A

MacromoleculeName: Transcription factor II-A / type: protein_or_peptide / ID: 2 / Name.synonym: TFIIA / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa / synonym: Human / Organelle: Nucleus
Molecular weightExperimental: 1.4 MDa / Theoretical: 1.4 MDa

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Macromolecule #3: Transcription factor SCP

MacromoleculeName: Transcription factor SCP / type: protein_or_peptide / ID: 3 / Name.synonym: SCP / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa / synonym: Human / Organelle: Nucleus
Molecular weightExperimental: 1.4 MDa / Theoretical: 1.4 MDa

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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

Concentration0.2 mg/mL
BufferpH: 7.8
Details: 20 mM HEPES pH 7.8, 0.2 mM EDTA, 4 mM MgCl2, ~1% glycerol, 1 mM DTT, 3% trehalose, 50 mM KCl, 0.05% NP-40,
GridDetails: 400 mesh copper grid on C-flat support carbon with a thin carbon support over 4 um holes space 2 um apart.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK II
Method: Sample was incubated on grid for 30 seconds before blotting 6.5s at -1 offset. The grid was washed in 4 ul buffer to blot again for 6.5 s at -1 offset prior to plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
Alignment procedureLegacy - Astigmatism: Objective and condenser lens astigmatism was corrected at 80,000 times magnification
DateMay 10, 2011
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 1037 / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 10500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 80000
Sample stageSpecimen holder: Gatan 626 liquid nitrogen cooled / Specimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles were prepared within the APPION processing environment. The particles were automatically selected using Signature and extracted from phase flipped micrographs using values calculated by CTFFIND3. After projection matching within EMAN2/Sparx, the final map was further refined in FREALIGN.
CTF correctionDetails: Per micrograph for phase flipping, per particles for refinement in FREALIGN
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Sparx, EMAN2, FREALIGN / Number images used: 8715

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