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- EMDB-48823: Structure of a native Drosophila melanogaster Nucleosome Elongati... -

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Basic information

Entry
Database: EMDB / ID: EMD-48823
TitleStructure of a native Drosophila melanogaster Nucleosome Elongation Complex (Pol II EC-nucleosome). Focused refinement of nucleosome
Map dataLocally refined, unsharpened nucleosome from the Nucleosome Elongation Complex, resolved to 6.12 A
Sample
  • Complex: Native purified Nucleosome Elongation Complex from Drosophila melanogaster
Keywordspolymerase / Pol II / transcription / mRNA
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.12 Å
AuthorsVenette-Smith NL / Vishwakarma RK / Dollinger R / Schultz J / Venkatakrishnan V / Babitzke P / Anand G / Gilmour DS / Armache J-P / Murakami K
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131860 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM047477 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM098399 United States
CitationJournal: To Be Published
Title: Structural Characterization of Native RNA Polymerase II Transcription Complexes in Drosophila melanogaster
Authors: Venette-Smith NL / Vishwakarma RK / Dollinger R / Schultz J / Venkatakrishnan V / Babitzke P / Anand G / Gilmour DS / Armache J-P / Murakami K
History
DepositionJan 27, 2025-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateFeb 19, 2025-
Current statusFeb 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48823.png

Downloads & links

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Map

FileDownload / File: emd_48823.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocally refined, unsharpened nucleosome from the Nucleosome Elongation Complex, resolved to 6.12 A
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 360 pix.
= 415.368 Å		1.15 Å/pix.
x 360 pix.
= 415.368 Å		1.15 Å/pix.
x 360 pix.
= 415.368 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1538 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.19035369 - 1.024966
Average (Standard dev.)0.0011008932 (±0.028258698)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 415.368 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Locally refined, sharpened Pol II from the Nucleosome...

Fileemd_48823_additional_1.map
AnnotationLocally refined, sharpened Pol II from the Nucleosome Elongation Complex, resolved to 6.12 A (bfactor = -385.9)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_48823_half_map_1.map
AnnotationHalf-map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2

Fileemd_48823_half_map_2.map
AnnotationHalf-map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Native purified Nucleosome Elongation Complex from Drosophila mel...

EntireName: Native purified Nucleosome Elongation Complex from Drosophila melanogaster
Components
  • Complex: Native purified Nucleosome Elongation Complex from Drosophila melanogaster

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Supramolecule #1: Native purified Nucleosome Elongation Complex from Drosophila mel...

SupramoleculeName: Native purified Nucleosome Elongation Complex from Drosophila melanogaster
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#15
Details: An overall structure of a native purified Nucleosome Elongation Complex (Pol II EC - nucleosome), where the Pol II and nucleosome are both at a lower resolution due to their motion in respect to each other
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 750 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

Concentration1.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
10.0 mMHEPES-HClHEPES
5.0 percentGlycerolGlycerol
1.0 mMEDTAEDTA
350.0 ug/mLFLAGFLAG peptide

Details: 10 mM HEPES-HCl (pH = 7.5), 150 mM NaCl, 5% glycerol, 1 mM EDTA, 350 ug/mL 3x FLAG peptide, 1/1000th protease inhibitor
GridModel: Quantifoil / Material: GOLD / Mesh: 200 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV
Detailsdouble FLAG-tagged Pol II subunit Rpb1 was used for purification of Pol II complexes

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Electron microscopy

MicroscopeTFS TALOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 4 / Number real images: 31103 / Average electron dose: 50.65 e/Å2
Details: Although the data was collected, motion-corrected and CTF estimated using the pixel size of 0.944, all the subsequent data processing was performed using pixel size of 1.1538. This was ...Details: Although the data was collected, motion-corrected and CTF estimated using the pixel size of 0.944, all the subsequent data processing was performed using pixel size of 1.1538. This was achieved by extracting particles in box size 440x440 and Fourier-cropping it to box size 360x360
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 4197404
Details: Initially, nearly 15 million particles were picked from micrographs. However, the number of particles cited here (4,197,404) is the starting number of particles after initial cleanup and 3D ...Details: Initially, nearly 15 million particles were picked from micrographs. However, the number of particles cited here (4,197,404) is the starting number of particles after initial cleanup and 3D classification of the data representing Pol II and nucleosome subsets, respectively.
Startup modelType of model: INSILICO MODEL / In silico model: 'Ab initio' function in cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.12 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1)
Details: The Nucleosome EC was refined to an overall resolution of 7.8 A. Then, local refinement was performed with an individual focus on the nucleosome and Pol II
Number images used: 36552
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.3.1) / Details: 'Non-Uniform Refinement' in cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementOverall B value: 50

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