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- EMDB-15858: Negative stain EM map of human RE1-silencing transcription factor -

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Basic information

Entry
Database: EMDB / ID: EMD-15858
TitleNegative stain EM map of human RE1-silencing transcription factor
Map dataREST negative staining EM map
Sample
  • Complex: RE1-silencing transcription factor
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 18.5 Å
AuthorsPinkas M / Brom T / Novacek J / Veverka P / Janovic T / Stojaspal M / Hofr C
Funding support Czech Republic, 3 items
OrganizationGrant numberCountry
Ministry of Education, Youth and Sports of the Czech RepublicLM2018127 Czech Republic
Czech Science Foundation19-18226S Czech Republic
Ministry of Education, Youth and Sports of the Czech RepublicLTAUSA19024 Czech Republic
CitationJournal: Comput Struct Biotechnol J / Year: 2023
Title: Electron microscopy reveals toroidal shape of master neuronal cell differentiator REST - RE1-silencing transcription factor.
Authors: Pavel Veverka / Tomáš Brom / Tomáš Janovič / Martin Stojaspal / Matyáš Pinkas / Jiří Nováček / Ctirad Hofr /
Abstract: The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map ...The RE1-Silencing Transcription factor (REST) is essential for neuronal differentiation. Here, we report the first 18.5-angstrom electron microscopy structure of human REST. The refined electron map suggests that REST forms a torus that can accommodate DNA double-helix in the central hole. Additionally, we quantitatively described REST binding to the canonical DNA sequence of the neuron-restrictive silencer element. We developed protocols for the expression and purification of full-length REST and the shortened variant REST-N62 produced by alternative splicing. We tested the mutual interaction of full-length REST and the splicing variant REST-N62. Revealed structure-function relationships of master neuronal repressor REST will allow finding new biological ways of prevention and treatment of neurodegenerative disorders and diseases.
History
DepositionSep 22, 2022-
Header (metadata) releaseDec 28, 2022-
Map releaseDec 28, 2022-
UpdateFeb 8, 2023-
Current statusFeb 8, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15858.png

Downloads & links

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Map

FileDownload / File: emd_15858.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationREST negative staining EM map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.65 Å/pix.
x 128 pix.
= 338.816 Å		2.65 Å/pix.
x 128 pix.
= 338.816 Å		2.65 Å/pix.
x 128 pix.
= 338.816 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.647 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.59
Minimum - Maximum-0.37291563 - 4.853586
Average (Standard dev.)0.028313309 (±0.19568415)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 338.816 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: REST half1 map

Fileemd_15858_half_map_1.map
AnnotationREST half1 map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: REST half2 map

Fileemd_15858_half_map_2.map
AnnotationREST half2 map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RE1-silencing transcription factor

EntireName: RE1-silencing transcription factor
Components
  • Complex: RE1-silencing transcription factor

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Supramolecule #1: RE1-silencing transcription factor

SupramoleculeName: RE1-silencing transcription factor / type: complex / ID: 1 / Chimera: Yes / Parent: 0
Source (natural)Organism: Homo sapiens (human) / Location in cell: nucleus, cytoplasm
Molecular weightTheoretical: 122 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMNaPisodium phosphate
150.0 mMNaClsodium chloride

Details: Buffer was prepared fresh then it was filtered by 0,22 um filter and degassed using ultrasound.
StainingType: NEGATIVE / Material: uranyl acetate
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 12.0 nm / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: OTHER

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Electron microscopy

MicroscopeTFS TALOS F200C
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Average electron dose: 15.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ab initio
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 18.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 3540
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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