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- EMDB-3455: Thermoplasma acidophilum 20S proteasome map reconstructed with Re... -

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Basic information

Entry
Database: EMDB / ID: EMD-3455
TitleThermoplasma acidophilum 20S proteasome map reconstructed with Relion 2.0 particle polishing from defocused Volta phase plate cryo-EM data
Map dataThermoplasma acidophilum 20S proteasome map reconstructed with Relion 2.0 particle polishing from defocused Volta phase plate cryo-EM data.
Sample
  • Complex: Thermoplasma acidophilum 20S proteasome
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsDanev R / Tegunov D / Baumeister W
CitationJournal: Elife / Year: 2017
Title: Using the Volta phase plate with defocus for cryo-EM single particle analysis.
Authors: Radostin Danev / Dimitry Tegunov / Wolfgang Baumeister /
Abstract: Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a ...Previously, we reported an in-focus data acquisition method for cryo-EM single-particle analysis with the Volta phase plate (Danev and Baumeister, 2016). Here, we extend the technique to include a small amount of defocus which enables contrast transfer function measurement and correction. This hybrid approach simplifies the experiment and increases the data acquisition speed. It also removes the resolution limit inherent to the in-focus method thus allowing 3D reconstructions with resolutions better than 3 Å.
History
DepositionNov 2, 2016-
Header (metadata) releaseNov 16, 2016-
Map releaseNov 16, 2016-
UpdateSep 30, 2020-
Current statusSep 30, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.14
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.14
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.14
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3455.png

Downloads & links

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Map

FileDownload / File: emd_3455.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThermoplasma acidophilum 20S proteasome map reconstructed with Relion 2.0 particle polishing from defocused Volta phase plate cryo-EM data.
Voxel sizeX=Y=Z: 0.527 Å
Density
Contour LevelBy AUTHOR: 0.14 / Movie #1: 0.14
Minimum - Maximum-0.24980377 - 0.44244894
Average (Standard dev.)0.00020806071 (±0.03128669)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 189.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5270.5270.527
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z189.720189.720189.720
α/β/γ90.00090.00090.000
start NX/NY/NZ212211153
NX/NY/NZ80102186
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.2500.4420.000

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Supplemental data

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Sample components

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Entire : Thermoplasma acidophilum 20S proteasome

EntireName: Thermoplasma acidophilum 20S proteasome
Components
  • Complex: Thermoplasma acidophilum 20S proteasome

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Supramolecule #1: Thermoplasma acidophilum 20S proteasome

SupramoleculeName: Thermoplasma acidophilum 20S proteasome / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Recombinant plasmid: pT7-7
Molecular weightTheoretical: 700 KDa

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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: 7.6 / Details: 25 mM Tris-HCL
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.1 kPa
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK III
Details: 3 ul of 0.5 mg/ml protein solution was applied on a grid in the Vitrobot chamber set to 95% RH at 20 degC then blotted for 5 s and plunged.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 0.7 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 47000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.62 mm / Nominal defocus max: 0.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 47000
Specialist opticsPhase plate: VOLTA PHASE PLATE / Energy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-24 / Number grids imaged: 1 / Number real images: 468 / Average exposure time: 12.0 sec. / Average electron dose: 39.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 145870
CTF correctionSoftware - Name: MATLAB / Software - details: homemade MATLAB program / Details: Relion 2.0 CTF correction
Startup modelType of model: EMDB MAP
EMDB ID:

3347


Details: 60 A low-pass filtered initial reference
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D7 (2x7 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 93596

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 74

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