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- EMDB-7154: Mtb 20S proteasome on a carbon nanowire grid plunged with Spotiton -

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Basic information

Entry
Database: EMDB / ID: EMD-7154
TitleMtb 20S proteasome on a carbon nanowire grid plunged with Spotiton
Map dataTomogram of Mtb 20S proteasome single particle
Sample
  • Complex: Mtb 20S proteasome
Biological speciesMycobacterium tuberculosis (bacteria)
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

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Map

FileDownload / File: emd_7154.map.gz / Format: CCP4 / Size: 1.3 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationTomogram of Mtb 20S proteasome single particle
Voxel sizeX=Y=Z: 1 Å
Density
Minimum - Maximum-0.028137034 - 0.05685799
Average (Standard dev.)0.014700062 (±0.0035118433)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin00-348
Dimensions9761006348
Spacing1006976348
CellA: 1006.0 Å / B: 976.0 Å / C: 348.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z1006976348
origin x/y/z0.0000.0000.000
length x/y/z1006.000976.000348.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS00-348
NC/NR/NS1006976348
D min/max/mean-0.0280.0570.015

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Supplemental data

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Sample components

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Entire : Mtb 20S proteasome

EntireName: Mtb 20S proteasome
Components
  • Complex: Mtb 20S proteasome

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Supramolecule #1: Mtb 20S proteasome

SupramoleculeName: Mtb 20S proteasome / type: complex / ID: 1 / Parent: 0 / Details: Tomography on single particle sample
Source (natural)Organism: Mycobacterium tuberculosis (bacteria)

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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 / Details: Spotiton.
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 QUANTUM (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: 32
DetailsAppion-Protomo fiducial-less tilt-series alignment

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