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- EMDB-7151: T20S proteasome on a holey carbon grid -

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Basic information

Entry
Database: EMDB / ID: EMD-7151
TitleT20S proteasome on a holey carbon grid
Map dataPhase plate tomogram 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
Funding support United States, 5 items
OrganizationGrant numberCountry
Simons Foundation349247 United States
National Institutes of Health/National Institute of General Medical SciencesGM103310 United States
NYSTAR United States
National Institutes of Health/Office of the DirectorS10 OD019994-01 United States
Agouron InstituteF00316 United States
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-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing 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_7151.png

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Map

FileDownload / File: emd_7151.map.gz / Format: CCP4 / Size: 1.1 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhase plate tomogram of T20S proteasome single particle
Voxel sizeX=Y=Z: 1 Å
Density
Minimum - Maximum-0.08687884 - 0.112583235
Average (Standard dev.)0.026657369 (±0.006940017)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-284
Dimensions10161046284
Spacing10461016284
CellA: 1046.0 Å / B: 1016.0 Å / C: 284.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z10461016284
origin x/y/z0.0000.0000.000
length x/y/z1046.0001016.000284.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-163-114-126
NX/NY/NZ210124170
MAP C/R/S123
start NC/NR/NS00-284
NC/NR/NS10461016284
D min/max/mean-0.0870.1130.027

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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
Specialist opticsPhase plate: VOLTA PHASE PLATE
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: 49
DetailsAppion-Protomo fiducial-less tilt-series alignment

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