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- EMDB-44650: Cryo-EM structure of Thermococcus kodakarensis FttA-dependent tra... -

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Basic information

Entry
Database: EMDB / ID: EMD-44650
TitleCryo-EM structure of Thermococcus kodakarensis FttA-dependent transcription pre-termination complex containing 52 nt RNA (local refinement map)
Map datalocal map
Sample
  • Complex: FttA-dependent transcription termination complex containing 52 nt RNA
KeywordsRNA polymerase / pre-termination complex / FttA / archaea / TRANSCRIPTION
Biological speciesThermococcus kodakarensis (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.34 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 29, 2024-
Header (metadata) releaseJul 31, 2024-
Map releaseJul 31, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44650.png

Downloads & links

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Map

FileDownload / File: emd_44650.map.gz / Format: CCP4 / Size: 352.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationlocal map
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 452 pix.
= 329.96 Å		0.73 Å/pix.
x 452 pix.
= 329.96 Å		0.73 Å/pix.
x 452 pix.
= 329.96 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.73 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.076
Minimum - Maximum-0.24009795 - 0.59975743
Average (Standard dev.)0.00040312237 (±0.012463542)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions452452452
Spacing452452452
CellA=B=C: 329.96002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44650_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_44650_half_map_2.map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : FttA-dependent transcription termination complex containing 52 nt RNA

EntireName: FttA-dependent transcription termination complex containing 52 nt RNA
Components
  • Complex: FttA-dependent transcription termination complex containing 52 nt RNA

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Supramolecule #1: FttA-dependent transcription termination complex containing 52 nt RNA

SupramoleculeName: FttA-dependent transcription termination complex containing 52 nt RNA
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 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 300 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 48.33 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: 2.0 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 165000
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: 977159
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.34 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 137835
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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