Ministry of Education, Youth and Sports of the Czech Republic
LM2018127
Czech Republic
Czech Science Foundation
19-18226S
Czech Republic
Ministry of Education, Youth and Sports of the Czech Republic
LTAUSA19024
Czech Republic
Citation
Journal: 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.
Organism: Homo sapiens (human) / Location in cell: nucleus, cytoplasm
Molecular weight
Theoretical: 122 KDa
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Experimental details
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Structure determination
Method
negative staining
Processing
single particle reconstruction
Aggregation state
particle
-
Sample preparation
Concentration
0.02 mg/mL
Buffer
pH: 8 Component:
Concentration
Formula
Name
50.0 mM
NaPi
sodium phosphate
150.0 mM
NaCl
sodium chloride
Details: Buffer was prepared fresh then it was filtered by 0,22 um filter and degassed using ultrasound.
Staining
Type: NEGATIVE / Material: uranyl acetate
Grid
Model: 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
Microscope
TFS TALOS F200C
Image recording
Film or detector model: FEI CETA (4k x 4k) / Average electron dose: 15.0 e/Å2
Electron beam
Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
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