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- EMDB-8999: Beam-tilt dependency of single-particle cryo-EM map quality: Expt... -

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Basic information

Entry
Database: EMDB / ID: EMD-8999
TitleBeam-tilt dependency of single-particle cryo-EM map quality: Expt 2 at 0 mrad
Map dataSharpened 0 beam tilt map in Experiment 2
Sample
  • Complex: Thermoplasma acidophilum 20S proteasome
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.74 Å
AuthorsCheng A / Eng ET / Alink L / Rice WJ / Jordan KD / Kim LY / Potter CS / Carragher B
CitationJournal: J Struct Biol / Year: 2018
Title: High resolution single particle cryo-electron microscopy using beam-image shift.
Authors: Anchi Cheng / Edward T Eng / Lambertus Alink / William J Rice / Kelsey D Jordan / Laura Y Kim / Clinton S Potter / Bridget Carragher /
Abstract: Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron ...Automated data acquisition is used widely for single-particle reconstruction of three-dimensional (3D) volumes of biological complexes preserved in vitreous ice and imaged in a transmission electron microscope. Automation has become integral to this method because of the very large number of particle images required in order to overcome the typically low signal-to-noise ratio of these images. For optimal efficiency, automated data acquisition software packages typically employ some beam-image shift targeting as this method is both fast and accurate (±0.1 µm). In contrast, using only stage movement, relocation to a targeted area under low-dose conditions can only be achieved in combination with multiple iterations or long relaxation times, both reducing efficiency. Nevertheless it is well known that applying beam-image shift induces beam-tilt and with it a potential structure phase error with a phase error π/4 the highest acceptable value. This theory has been used as an argument against beam-image shift for high resolution data collection. Nevertheless, in practice many small beam-image shift datasets have resulted in 3D reconstructions beyond the π/4 phase error limit. To address this apparent contradiction, we performed cryo-EM single-particle reconstructions on a T20S proteasome sample using applied beam-image shifts corresponding to beam tilts from 0 to 10 mrad. To evaluate the results we compared the FSC values, and examined the water density peaks in the 3D map. We conclude that the phase error does not limit the validity of the 3D reconstruction from single-particle averaging beyond the π/4 resolution limit.
History
DepositionJul 26, 2018-
Header (metadata) releaseAug 8, 2018-
Map releaseAug 22, 2018-
UpdateOct 10, 2018-
Current statusOct 10, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.05
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.05
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8999.png

Downloads & links

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Map

FileDownload / File: emd_8999.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened 0 beam tilt map in Experiment 2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.488 Å		1.06 Å/pix.
x 256 pix.
= 271.488 Å		1.06 Å/pix.
x 256 pix.
= 271.488 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0605 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.05 / Movie #1: 1.05
Minimum - Maximum-4.1709642 - 6.7849107
Average (Standard dev.)0.0019432057 (±0.31377828)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.488 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06051.06051.0605
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.488271.488271.488
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-4.1716.7850.002

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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)
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

Concentration0.9 mg/mL
BufferpH: 8 / Component:
ConcentrationNameFormula
20.0 mMTris
150.0 mMNaCl
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 75 % / Chamber temperature: 298 K / Instrument: GATAN CRYOPLUNGE 3 / Details: blotted for 2.5 s after 30 s wait time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 15 eV
Details0,0 beam-image shift with Intentionally beam-shift pivot misalignment.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 2-60 / Number grids imaged: 1 / Number real images: 545 / Average exposure time: 6.0 sec. / Average electron dose: 0.71 e/Å2
Details: of the 545 images, 158 images were removed from processing because of empty or high drift movie.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: NONE / Details: CryoSPARC ab initio
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D7 (2x7 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.74 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 0.6.5) / Number images used: 11400
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 0.6.5)
Final angle assignmentType: MAXIMUM LIKELIHOOD

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