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- EMDB-7153: T20S proteasome on a gold Quantifoil grid -

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Basic information

Entry
Database: EMDB / ID: EMD-7153
TitleT20S proteasome on a gold Quantifoil grid
Map dataTomogram of T20S proteasome single particle
Sample
  • Complex: T20S proteasome
Biological speciesunidentified (others)
Methodelectron tomography / cryo EM
AuthorsNoble AN / Dandey VP / Wei H / Brasch J / Chase J / Acharya P / Tan Y / Zhang Z / Kim LY / Scapin G ...Noble AN / Dandey VP / Wei H / Brasch J / Chase J / Acharya P / Tan Y / Zhang Z / Kim LY / Scapin G / Rapp M / Eng ET / Rice WJ / Cheng A / Negro CJ / Shapiro L / Kwong PD / Jeruzalmi D / des Georges A / Potter CS / Carragher B
CitationJournal: Elife / Year: 2018
Title: Routine single particle CryoEM sample and grid characterization by tomography.
Authors: Alex J Noble / Venkata P Dandey / Hui Wei / Julia Brasch / Jillian Chase / Priyamvada Acharya / Yong Zi Tan / Zhening Zhang / Laura Y Kim / Giovanna Scapin / Micah Rapp / Edward T Eng / ...Authors: Alex J Noble / Venkata P Dandey / Hui Wei / Julia Brasch / Jillian Chase / Priyamvada Acharya / Yong Zi Tan / Zhening Zhang / Laura Y Kim / Giovanna Scapin / Micah Rapp / Edward T Eng / William J Rice / Anchi Cheng / Carl J Negro / Lawrence Shapiro / Peter D Kwong / David Jeruzalmi / Amedee des Georges / Clinton S Potter / Bridget Carragher /
Abstract: Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we ...Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are not adsorbed to the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.
History
DepositionDec 5, 2017-
Header (metadata) releaseDec 20, 2017-
Map releaseDec 20, 2017-
UpdateJun 13, 2018-
Current statusJun 13, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • 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_7153.png

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Map

FileDownload / File: emd_7153.map.gz / Format: CCP4 / Size: 1.7 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of T20S proteasome single particle
Voxel sizeX=Y=Z: 1 Å
Density
Minimum - Maximum-0.2642808 - 0.11876096
Average (Standard dev.)0.030521635 (±0.0054742955)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-426
Dimensions10161044426
Spacing10441016426
CellA: 1044.0 Å / B: 1016.0 Å / C: 426.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z10441016426
origin x/y/z0.0000.0000.000
length x/y/z1044.0001016.000426.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00-426
NC/NR/NS10441016426
D min/max/mean-0.2640.1190.031

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Supplemental data

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Sample components

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Entire : T20S proteasome

EntireName: T20S proteasome
Components
  • Complex: T20S proteasome

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Supramolecule #1: T20S proteasome

SupramoleculeName: T20S proteasome / type: complex / ID: 1 / Parent: 0 / Details: Tomography on single particle sample
Source (natural)Organism: unidentified (others)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

VitrificationCryogen name: ETHANE
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 2.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: SIMULTANEOUS ITERATIVE (SIRT) / Software - Name: TOMO3D / Number images used: 28
DetailsAppion-Protomo fiducial-less tilt-series alignment

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