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- EMDB-48624: Structure of a native Drosophila melanogaster Pol II Elongation C... -

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Entry
Database: EMDB / ID: EMD-48624
TitleStructure of a native Drosophila melanogaster Pol II Elongation Complex
Map dataRefined, unsharpened Coulomb potential density map. Particles extracted in 440x440 Fourier cropped to 360x360.
Sample
  • Complex: Natively purified Pol II elongation complex from Drosophila melanogaster. This entry contains a Coulomb potential density map which is a mix of polymerases with and without stalk.
Keywordspolymerase / Pol II / transcription / mRNA
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.86 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: Nat Commun / Year: 2026
Title: Structural Characterization of Native RNA Polymerase II Transcription Complexes and Nucleosomes in Drosophila melanogaster.
Authors: Natalie L Venette-Smith / Rishi K Vishwakarma / Varun Venkatakrishnan / Roberta Dollinger / Josie Schultz / Paul Babitzke / Ganesh Anand / David S Gilmour / Jean-Paul Armache / Katsuhiko S Murakami /
Abstract: Structural studies of eukaryotic RNA polymerase II (Pol II) transcription often rely on in vitro assembly, which may not fully represent native conditions. To investigate Pol II transcription in ...Structural studies of eukaryotic RNA polymerase II (Pol II) transcription often rely on in vitro assembly, which may not fully represent native conditions. To investigate Pol II transcription in metazoan cells, we developed a method to isolate native transcription complexes from Drosophila melanogaster embryos using FLAG-tag affinity purification and Micrococcal Nuclease treatment. Cryo-EM and proteomics studies revealed diverse transcription complexes and nucleosomes, including a metazoan Rpb4/Rpb7 stalk-less Pol II elongation complex and a hexameric nucleosome lacking an H2A/H2B dimer. Notably, nucleosome is found only downstream of the nucleosome elongation complex, underscoring it as a major energy barrier and a time-consuming step during Pol II progression through chromatin. Proteomics identified co-purified factors involved in transcription initiation, elongation, and RNA modification. This study provides a framework for investigations of transcription in cells, paving the way for future studies of transient and minor complexes.
History
DepositionJan 13, 2025-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateSep 30, 2026-
Current statusSep 30, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48624.png

Downloads & links

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Map

FileDownload / File: emd_48624.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRefined, unsharpened Coulomb potential density map. Particles extracted in 440x440 Fourier cropped to 360x360.
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: 0.4
Minimum - Maximum-0.0 - 2.7697618
Average (Standard dev.)0.011336053 (±0.061930116)
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: Refined, auto-sharpened Coulomb potential density map with Bfactor...

Fileemd_48624_additional_1.map
AnnotationRefined, auto-sharpened Coulomb potential density map with Bfactor = -47.8
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1

Fileemd_48624_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_48624_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 : Natively purified Pol II elongation complex from Drosophila melan...

EntireName: Natively purified Pol II elongation complex from Drosophila melanogaster. This entry contains a Coulomb potential density map which is a mix of polymerases with and without stalk.
Components
  • Complex: Natively purified Pol II elongation complex from Drosophila melanogaster. This entry contains a Coulomb potential density map which is a mix of polymerases with and without stalk.

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Supramolecule #1: Natively purified Pol II elongation complex from Drosophila melan...

SupramoleculeName: Natively purified Pol II elongation complex from Drosophila melanogaster. This entry contains a Coulomb potential density map which is a mix of polymerases with and without stalk.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#15
Details: This entry represents a native Pol II elongation complex which is the best-resolved overall reconstruction from this dataset. It is composed of particles with Rpb4/Rpb7 stalk, as well as without Rpb4/Rpb7 stalk
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 930 kDa/nm

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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: 2349308
Details: Initially, nearly 15 million particles were picked from micrographs. However, the number of particles cited here (2,349,308) 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 (2,349,308) is the starting number of particles after initial cleanup and 3D classification of the data.
CTF correctionSoftware - Name: cryoSPARC (ver. 4.3.1) / Details: 'Patch CTF Estimation' in cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
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: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.3.1)
Details: The data was processed using cryoSPARC to 2.96 A from 876,764 particles. It was later post-processed using CryoSieve to select the final number of particles, yielding 2.86 A.
Number images used: 68716
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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