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- EMDB-44454: Cryo-EM structure of Thermococcus kodakarensis FttA-dependent tra... -

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Entry
Database: EMDB / ID: EMD-44454
TitleCryo-EM structure of Thermococcus kodakarensis FttA-dependent transcription pre-termination complex containing 44 nt RNA
Map dataoverall map
Sample
  • Complex: FttA-dependent transcription termination complex
KeywordsRNA polymerase / pre-termination complex / FttA / archaea / TRANSCRIPTION
Biological speciesThermococcus kodakarensis (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsYou L / Ebright RH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nature / Year: 2024
Title: Structural basis of archaeal FttA-dependent transcription termination.
Authors: Linlin You / Chengyuan Wang / Vadim Molodtsov / Konstantin Kuznedelov / Xinyi Miao / Breanna R Wenck / Paul Ulisse / Travis J Sanders / Craig J Marshall / Emre Firlar / Jason T Kaelber / ...Authors: Linlin You / Chengyuan Wang / Vadim Molodtsov / Konstantin Kuznedelov / Xinyi Miao / Breanna R Wenck / Paul Ulisse / Travis J Sanders / Craig J Marshall / Emre Firlar / Jason T Kaelber / Thomas J Santangelo / Richard H Ebright /
Abstract: The ribonuclease FttA (also known as aCPSF and aCPSF1) mediates factor-dependent transcription termination in archaea. Here we report the structure of a Thermococcus kodakarensis transcription pre- ...The ribonuclease FttA (also known as aCPSF and aCPSF1) mediates factor-dependent transcription termination in archaea. Here we report the structure of a Thermococcus kodakarensis transcription pre-termination complex comprising FttA, Spt4, Spt5 and a transcription elongation complex (TEC). The structure shows that FttA interacts with the TEC in a manner that enables RNA to proceed directly from the TEC RNA-exit channel to the FttA catalytic centre and that enables endonucleolytic cleavage of RNA by FttA, followed by 5'→3' exonucleolytic cleavage of RNA by FttA and concomitant 5'→3' translocation of FttA on RNA, to apply mechanical force to the TEC and trigger termination. The structure further reveals that Spt5 bridges FttA and the TEC, explaining how Spt5 stimulates FttA-dependent termination. The results reveal functional analogy between bacterial and archaeal factor-dependent termination, functional homology between archaeal and eukaryotic factor-dependent termination, and fundamental mechanistic similarities in factor-dependent termination in bacteria, archaea, and eukaryotes.
History
DepositionApr 12, 2024-
Header (metadata) releaseJul 31, 2024-
Map releaseJul 31, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44454.png

Downloads & links

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Map

FileDownload / File: emd_44454.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationoverall map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 330. Å		0.83 Å/pix.
x 400 pix.
= 330. Å		0.83 Å/pix.
x 400 pix.
= 330. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.082
Minimum - Maximum-0.26518983 - 0.6446787
Average (Standard dev.)0.001195497 (±0.01906338)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 330.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44454_msk_1.map
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Additional map: local map

Fileemd_44454_additional_1.map
Annotationlocal map
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Half map: #2

Fileemd_44454_half_map_1.map
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Half map: #1

Fileemd_44454_half_map_2.map
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Sample components

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Entire : FttA-dependent transcription termination complex

EntireName: FttA-dependent transcription termination complex
Components
  • Complex: FttA-dependent transcription termination complex

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Supramolecule #1: FttA-dependent transcription termination complex

SupramoleculeName: FttA-dependent transcription termination complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#17
Source (natural)Organism: Thermococcus kodakarensis (archaea)
Molecular weightTheoretical: 556 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

Concentration4.5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 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: 2.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
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

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 171269
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT

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