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- EMDB-4305: Correlative cryo-FM and Cryo-ET of vitreous sections of yeast cells -

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Basic information

Entry
Database: EMDB / ID: EMD-4305
TitleCorrelative cryo-FM and Cryo-ET of vitreous sections of yeast cells
Map dataThis is a tomogram of a vitreous section of yeast cells expressing Pil1-GFP. There are two eisosomal sites seen.
Sample
  • Cell: Saccharomyces cerevisiae
Biological speciesSaccharomyces cerevisiae S288c (yeast)
Methodelectron tomography
AuthorsBharat TAM / Hoffmann PC / Kukulski W
CitationJournal: Structure / Year: 2018
Title: Correlative Microscopy of Vitreous Sections Provides Insights into BAR-Domain Organization In Situ.
Authors: Tanmay A M Bharat / Patrick C Hoffmann / Wanda Kukulski /
Abstract: Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to ...Electron microscopy imaging of macromolecular complexes in their native cellular context is limited by the inherent difficulty to acquire high-resolution tomographic data from thick cells and to specifically identify elusive structures within crowded cellular environments. Here, we combined cryo-fluorescence microscopy with electron cryo-tomography of vitreous sections into a coherent correlative microscopy workflow, ideal for detection and structural analysis of elusive protein assemblies in situ. We used this workflow to address an open question on BAR-domain coating of yeast plasma membrane compartments known as eisosomes. BAR domains can sense or induce membrane curvature, and form scaffold-like membrane coats in vitro. Our results demonstrate that in cells, the BAR protein Pil1 localizes to eisosomes of varying membrane curvature. Sub-tomogram analysis revealed a dense protein coat on curved eisosomes, which was not present on shallow eisosomes, indicating that while BAR domains can assemble at shallow membranes in vivo, scaffold formation is tightly coupled to curvature generation.
History
DepositionFeb 21, 2018-
Header (metadata) releaseMay 2, 2018-
Map releaseMay 2, 2018-
UpdateJun 13, 2018-
Current statusJun 13, 2018Processing site: PDBe / Status: Released

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Structure visualization

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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Supplemental images

emd_4305.png

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Map

FileDownload / File: emd_4305.map.gz / Format: CCP4 / Size: 221.7 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationThis is a tomogram of a vitreous section of yeast cells expressing Pil1-GFP. There are two eisosomal sites seen.
Voxel sizeX=Y=Z: 14.34 Å
Density
Minimum - Maximum-128. - 127.
Average (Standard dev.)64.033810000000003 (±97.644189999999995)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-1616151
Dimensions926958262
Spacing958926262
CellA: 13737.72 Å / B: 13278.84 Å / C: 3757.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z14.3414.3414.34
M x/y/z958926262
origin x/y/z0.0000.0000.000
length x/y/z13737.72013278.8403757.080
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS16-16151
NC/NR/NS958926262
D min/max/mean-128.000127.00064.034

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Supplemental data

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Sample components

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Entire : Saccharomyces cerevisiae

EntireName: Saccharomyces cerevisiae (brewer's yeast)
Components
  • Cell: Saccharomyces cerevisiae

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Supramolecule #1: Saccharomyces cerevisiae

SupramoleculeName: Saccharomyces cerevisiae / type: cell / ID: 1 / Parent: 0
Details: genotype: MATalpha, his3delta200, leu2-3,112, ura3-52, lys2-801, PIL1-EGFP::HIS3MX6
Source (natural)Organism: Saccharomyces cerevisiae S288c (yeast)

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Experimental details

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Structure determination

Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
High pressure freezingInstrument: OTHER
Details: The value given for _emd_high_pressure_freezing.instrument is Leica HPM100. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER', ...Details: The value given for _emd_high_pressure_freezing.instrument is Leica HPM100. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION FREEZER', 'OTHER', 'LEICA EM HPM100', 'BAL-TEC HPM 010']) so OTHER is written into the XML file.
SectioningUltramicrotomy - Instrument: Leica / Ultramicrotomy - Temperature: 100 K / Ultramicrotomy - Final thickness: 150
Fiducial markerManufacturer: CMC Utrecht / Diameter: 10 nm

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 0.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: IMOD / Number images used: 121
CTF correctionSoftware - Name: IMOD

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