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- EMDB-4184: RNA Polymerase III pre-initiation complex -

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Basic information

Entry
Database: EMDB / ID: EMD-4184
TitleRNA Polymerase III pre-initiation complex
Map dataMap of the RNA Pol III pre-initiation complex
Sample
  • Complex: RNA Polymerase III pre-initiation complex
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsVorlaender MK / Khatter H / Wetzel R / Hagen WJ / Mueller CW
CitationJournal: Nature / Year: 2018
Title: Molecular mechanism of promoter opening by RNA polymerase III.
Authors: Matthias K Vorländer / Heena Khatter / Rene Wetzel / Wim J H Hagen / Christoph W Müller /
Abstract: RNA polymerase III (Pol III) and transcription factor IIIB (TFIIIB) assemble together on different promoter types to initiate the transcription of small, structured RNAs. Here we present structures ...RNA polymerase III (Pol III) and transcription factor IIIB (TFIIIB) assemble together on different promoter types to initiate the transcription of small, structured RNAs. Here we present structures of Pol III preinitiation complexes, comprising the 17-subunit Pol III and the heterotrimeric transcription factor TFIIIB, bound to a natural promoter in different functional states. Electron cryo-microscopy reconstructions, varying from 3.7 Å to 5.5 Å resolution, include two early intermediates in which the DNA duplex is closed, an open DNA complex, and an initially transcribing complex with RNA in the active site. Our structures reveal an extremely tight, multivalent interaction between TFIIIB and promoter DNA, and explain how TFIIIB recruits Pol III. Together, TFIIIB and Pol III subunit C37 activate the intrinsic transcription factor-like activity of the Pol III-specific heterotrimer to initiate the melting of double-stranded DNA, in a mechanism similar to that of the Pol II system.
History
DepositionNov 29, 2017-
Header (metadata) releaseJan 17, 2018-
Map releaseJan 31, 2018-
UpdateJan 31, 2018-
Current statusJan 31, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4184.png

Downloads & links

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Map

FileDownload / File: emd_4184.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of the RNA Pol III pre-initiation complex
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 3. / Movie #1: 3
Minimum - Maximum-11.47481 - 18.611363999999998
Average (Standard dev.)0.018354587 (±0.7109673)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 324.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z324.000324.000324.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ240240240
MAP C/R/S312
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-11.47518.6110.018

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Supplemental data

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Sample components

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Entire : RNA Polymerase III pre-initiation complex

EntireName: RNA Polymerase III pre-initiation complex
Components
  • Complex: RNA Polymerase III pre-initiation complex

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Supramolecule #1: RNA Polymerase III pre-initiation complex

SupramoleculeName: RNA Polymerase III pre-initiation complex / type: complex / ID: 1 / Parent: 0
Details: Map obtained before seperation into open DNA complex (OC) and closed DNA complex (CC)
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 893 KDa

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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

Concentration0.02 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsCrosslinked with glutaraldehyde

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 60.9 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5fj8

Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 62751
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 110 / Target criteria: Cross-correlation coefficient

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