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- EMDB-43502: Cryo-Electron Tomography of Wildtype LAF-1 RGG Domain Condensate ... -

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Basic information

Entry
Database: EMDB / ID: EMD-43502
TitleCryo-Electron Tomography of Wildtype LAF-1 RGG Domain Condensate with Core/Shell Structure
Map dataCryo-electron tomogram of wildtype LAF-1 RGG condensate with core-shell structure
Sample
  • Complex: Wildtype LAF-1 RGG condensate
Keywordsbiomolecular condensate / phase separation / LLPS / intrinsic disorder / fibers / RNA BINDING PROTEIN
Biological speciesCaenorhabditis elegans (invertebrata)
Methodelectron tomography / cryo EM
AuthorsJaber N / Dai W / Schuster BS / Patterson JP
Funding support United States, 3 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DMR-2011967 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM142903 United States
National Science Foundation (NSF, United States)MCB-2046180 United States
CitationJournal: Nanoscale / Year: 2024
Title: Revealing nanoscale structure and interfaces of protein and polymer condensates cryo-electron microscopy.
Authors: Aoon Rizvi / Bruna Favetta / Nora Jaber / Yun-Kyung Lee / Jennifer Jiang / Nehal S Idris / Benjamin S Schuster / Wei Dai / Joseph P Patterson /
Abstract: Liquid-liquid phase separation (LLPS) is a ubiquitous demixing phenomenon observed in various molecular solutions, including in polymer and protein solutions. Demixing of solutions results in ...Liquid-liquid phase separation (LLPS) is a ubiquitous demixing phenomenon observed in various molecular solutions, including in polymer and protein solutions. Demixing of solutions results in condensed, phase separated droplets which exhibit a range of liquid-like properties driven by transient intermolecular interactions. Understanding the organization within these condensates is crucial for deciphering their material properties and functions. This study explores the distinct nanoscale networks and interfaces in the condensate samples using a modified cryo-electron microscopy (cryo-EM) method. The method involves initiating condensate formation on electron microscopy grids to limit droplet growth as large droplet sizes are not ideal for cryo-EM imaging. The versatility of this method is demonstrated by imaging three different classes of condensates. We further investigate the condensate structures using cryo-electron tomography which provides 3D reconstructions, uncovering porous internal structures, unique core-shell morphologies, and inhomogeneities within the nanoscale organization of protein condensates. Comparison with dry-state transmission electron microscopy emphasizes the importance of preserving the hydrated structure of condensates for accurate structural analysis. We correlate the internal structure of protein condensates with their amino acid sequences and material properties by performing viscosity measurements that support that more viscous condensates exhibit denser internal assemblies. Our findings contribute to a comprehensive understanding of nanoscale condensate structure and its material properties. Our approach here provides a versatile tool for exploring various phase-separated systems and their nanoscale structures for future studies.
History
DepositionJan 24, 2024-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43502.png

Downloads & links

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Map

FileDownload / File: emd_43502.map.gz / Format: CCP4 / Size: 42.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-electron tomogram of wildtype LAF-1 RGG condensate with core-shell structure
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
17.2 Å/pix.
x 91 pix.
= 1565.109 Å		17.2 Å/pix.
x 328 pix.
= 5641.272 Å		17.2 Å/pix.
x 376 pix.
= 6466.824 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 17.199 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-9.997506 - 24.398775000000001
Average (Standard dev.)-0.15820804 (±1.2406127)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-410-301-89
Dimensions32837691
Spacing37632891
CellA: 6466.8237 Å / B: 5641.272 Å / C: 1565.1089 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Wildtype LAF-1 RGG condensate

EntireName: Wildtype LAF-1 RGG condensate
Components
  • Complex: Wildtype LAF-1 RGG condensate

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Supramolecule #1: Wildtype LAF-1 RGG condensate

SupramoleculeName: Wildtype LAF-1 RGG condensate / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Caenorhabditis elegans (invertebrata)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293.15 K / Instrument: LEICA EM GP / Details: Blot time: 4 seconds.
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: EMS / Diameter: 6 nm

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number real images: 41 / Average electron dose: 1.5 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 10.0 µm / Nominal defocus min: 8.0 µm / Nominal magnification: 31000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: EMAN2 / Number images used: 41

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