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- EMDB-10302: CLEM of resin-embedded scs2/22 ist2 deletion mutant yeast cell ex... -

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Basic information

Entry
Database: EMDB / ID: EMD-10302
TitleCLEM of resin-embedded scs2/22 ist2 deletion mutant yeast cell expressing Tcb3-GFP; shown in Figure 2G of publication
Map dataCLEM of resin-embedded scs2/22 ist2 deletion mutant yeast cell expressing Tcb3-GFP shown in Figure 2G of publication
Sample
  • Cell: Correlative FM and ET of resin-embedded scs2/22 ist2 deletion mutant yeast cell expressing Tcb3-GFP
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM / negative staining
AuthorsHoffmann PC / Bharat TAM / Wozny MR / Boulanger J / Miller EA / Kukulski W
Funding support United Kingdom, 3 items
OrganizationGrant numberCountry
Medical Research Council (United Kingdom)MC_UP_1201/8 United Kingdom
Wellcome Trust202231/Z/16/Z United Kingdom
Medical Research Council (United Kingdom)MC_UP_1201/10 United Kingdom
CitationJournal: Dev Cell / Year: 2019
Title: Tricalbins Contribute to Cellular Lipid Flux and Form Curved ER-PM Contacts that Are Bridged by Rod-Shaped Structures.
Authors: Patrick C Hoffmann / Tanmay A M Bharat / Michael R Wozny / Jerome Boulanger / Elizabeth A Miller / Wanda Kukulski /
Abstract: Lipid flow between cellular organelles occurs via membrane contact sites. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and ...Lipid flow between cellular organelles occurs via membrane contact sites. Extended-synaptotagmins, known as tricalbins in yeast, mediate lipid transfer between the endoplasmic reticulum (ER) and plasma membrane (PM). How these proteins regulate membrane architecture to transport lipids across the aqueous space between bilayers remains unknown. Using correlative microscopy, electron cryo-tomography, and high-throughput genetics, we address the interplay of architecture and function in budding yeast. We find that ER-PM contacts differ in protein composition and membrane morphology, not in intermembrane distance. In situ electron cryo-tomography reveals the molecular organization of tricalbin-mediated contacts, suggesting a structural framework for putative lipid transfer. Genetic analysis uncovers functional overlap with cellular lipid routes, such as maintenance of PM asymmetry. Further redundancies are suggested for individual tricalbin protein domains. We propose a modularity of molecular and structural functions of tricalbins and of their roles within the cellular network of lipid distribution pathways.
History
DepositionSep 9, 2019-
Header (metadata) releaseNov 13, 2019-
Map releaseNov 13, 2019-
UpdateDec 4, 2019-
Current statusDec 4, 2019Processing 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_10302.png

Downloads & links

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Map

FileDownload / File: emd_10302.map.gz / Format: CCP4 / Size: 672 MB / Type: IMAGE STORED AS SIGNED BYTE
AnnotationCLEM of resin-embedded scs2/22 ist2 deletion mutant yeast cell expressing Tcb3-GFP shown in Figure 2G of publication
Voxel sizeX=Y=Z: 11.02 Å
Density
Minimum - Maximum-128 - 127
Average (Standard dev.)38.718597 (±7.0233808)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-84
Dimensions20482048168
Spacing20482048168
CellA: 22568.96 Å / B: 22568.96 Å / C: 1851.3601 Å
α=β=γ: 90.0 °

CCP4 map header:

modeenvelope stored as signed bytes (from -128 lowest to 127 highest)
Å/pix. X/Y/Z11.02000048828111.02000048828111.02
M x/y/z20482048168
origin x/y/z0.0000.0000.000
length x/y/z22568.96122568.9611851.360
α/β/γ90.00090.00090.000
start NX/NY/NZ111-94150
NX/NY/NZ111123111
MAP C/R/S123
start NC/NR/NS00-84
NC/NR/NS20482048168
D min/max/mean-128.000127.00038.719

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Supplemental data

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Sample components

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Entire : Correlative FM and ET of resin-embedded scs2/22 ist2 deletion mut...

EntireName: Correlative FM and ET of resin-embedded scs2/22 ist2 deletion mutant yeast cell expressing Tcb3-GFP
Components
  • Cell: Correlative FM and ET of resin-embedded scs2/22 ist2 deletion mutant yeast cell expressing Tcb3-GFP

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Supramolecule #1: Correlative FM and ET of resin-embedded scs2/22 ist2 deletion mut...

SupramoleculeName: Correlative FM and ET of resin-embedded scs2/22 ist2 deletion mutant yeast cell expressing Tcb3-GFP
type: cell / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingelectron tomography
Aggregation statecell

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Sample preparation

BufferpH: 7.4
StainingType: NONE / Material: Uranyl Acetate
Details: 0.03-0.05 % uranyl acetate during freeze substitution and post-staining of sections with Reynold's lead citrate
Sugar embeddingMaterial: Lowicryl HM20
VitrificationCryogen name: NITROGEN
High pressure freezingInstrument: OTHER
Details: The value given for _emd_high_pressure_freezing.instrument is Leica EM HPM100. This is not in a list of allowed values set(['LEICA EM PACT2', 'LEICA EM PACT', 'EMS-002 RAPID IMMERSION ...Details: The value given for _emd_high_pressure_freezing.instrument is Leica EM 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: Reichert Ultracut E / Ultramicrotomy - Temperature: 298 K / Ultramicrotomy - Final thickness: 270 nm
Fiducial marker
ManufacturerDiameter
Electron Microscopy Sciences15 nm
Life Technologies50 nm

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
DetailsSTEM mode on an axial brightfield detector with a high-tilt tomography holder (Fischione Instruments, Model 2020)
Image recordingFilm or detector model: OTHER / Average electron dose: 1000.0 e/Å2
Details: ET was done in STEM mode on an axial brightfield detector with a high-tilt tomography holder at 1.102 nm pixel size with a camera length of 200 mm. [NOTE: Electron dose unknown. Value specified here is a dummy]
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: eTomo
Final reconstructionSoftware - Name: eTomo / Details: 246 tilted images from two axes / Number images used: 246

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