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- EMDB-3348: cryo-EM structure of T20S proteasome -

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Basic information

Entry
Database: EMDB / ID: EMD-3348
Titlecryo-EM structure of T20S proteasome
Map datacryo-EM reconstruction of T20S proteasome
Sample
  • Sample: Thermoplasma acidophilum 20S proteasome
  • Protein or peptide: 20S proteasome
Keywordsproteasome / 20S
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsDanev R / Baumeister W
CitationJournal: Elife / Year: 2016
Title: Cryo-EM single particle analysis with the Volta phase plate.
Authors: Radostin Danev / Wolfgang Baumeister /
Abstract: We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining ...We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional defocus approach. Spherical aberration becomes a limiting factor for achieving resolutions below 3 Å with in-focus phase plate images. The phase plate could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach.
History
DepositionFeb 25, 2016-
Header (metadata) releaseMar 2, 2016-
Map releaseMar 16, 2016-
UpdateMar 16, 2016-
Current statusMar 16, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.11
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3348.png

Downloads & links

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Map

FileDownload / File: emd_3348.map.gz / Format: CCP4 / Size: 20.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcryo-EM reconstruction of T20S proteasome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 176 pix.
= 237.6 Å		1.35 Å/pix.
x 176 pix.
= 237.6 Å		1.35 Å/pix.
x 176 pix.
= 237.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.11 / Movie #1: 0.11
Minimum - Maximum-0.29501361 - 0.50774992
Average (Standard dev.)0.00008575 (±0.02720418)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions176176176
Spacing176176176
CellA=B=C: 237.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z176176176
origin x/y/z0.0000.0000.000
length x/y/z237.600237.600237.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-64
NX/NY/NZ6161129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS176176176
D min/max/mean-0.2950.5080.000

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Supplemental data

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Segmentation: the mask was automatically generated by Relion during postprocessing.

Annotationthe mask was automatically generated by Relion during postprocessing.
Fileemd_3348_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Thermoplasma acidophilum 20S proteasome

EntireName: Thermoplasma acidophilum 20S proteasome
Components
  • Sample: Thermoplasma acidophilum 20S proteasome
  • Protein or peptide: 20S proteasome

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Supramolecule #1000: Thermoplasma acidophilum 20S proteasome

SupramoleculeName: Thermoplasma acidophilum 20S proteasome / type: sample / ID: 1000 / Oligomeric state: 28-mer / Number unique components: 1
Molecular weightTheoretical: 700 KDa

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Macromolecule #1: 20S proteasome

MacromoleculeName: 20S proteasome / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: 28-mer / Recombinant expression: Yes
Source (natural)Organism: Thermoplasma acidophilum (acidophilic) / synonym: Thermoplasma
Molecular weightTheoretical: 700 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.5 mg/mL
BufferpH: 7 / Details: 25 mM Tris-HCl
GridDetails: 200 mesh Copper Quantifoil R 1.2/1.3
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 95 % / Chamber temperature: 80 K / Instrument: FEI VITROBOT MARK III
Method: 3 ul of sample, blot time 5 s, chamber temperature 20 degC

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureAverage: 80 K
Alignment procedureLegacy - Astigmatism: astigmatism was corrected manually at the magnification used for data acquisition.
Specialist opticsEnergy filter - Name: Gatan Quantum / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 10.0 eV
DateJul 21, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Digitization - Sampling interval: 5 µm / Number real images: 293 / Average electron dose: 30 e/Å2
Details: every image is the average of 12 frames on the direct detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 37037 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 37000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Detailsthe particles were semi-automatically selected with EMAN2 e2boxer
CTF correctionDetails: each micrograph
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: EMAN2, Relion, ctffind4 / Number images used: 35717
FSC plot (resolution estimation)

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