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- EMDB-8742: Thermoplasma acidophilum 20S Proteasome using 200keV with image shift -

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Basic information

Entry
Database: EMDB / ID: EMD-8742
TitleThermoplasma acidophilum 20S Proteasome using 200keV with image shift
Map dataFinal sharpened map of T. acidophilum 20S proteasome collected using image shift navigation
Sample
  • Complex: Thermoplasma acidophilum 20S proteasome
    • Protein or peptide: Proteasome subunit alpha
    • Protein or peptide: Proteasome subunit beta
KeywordsProteasome / hydrolase
Function / homology
Function and homology information


proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / proteasome core complex, alpha-subunit complex / threonine-type endopeptidase activity / proteasomal protein catabolic process / ubiquitin-dependent protein catabolic process / endopeptidase activity / cytoplasm
Similarity search - Function
Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / : ...Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Proteasome beta-type subunits signature. / Peptidase T1A, proteasome beta-subunit / Proteasome beta-type subunit, conserved site / Proteasome subunit A N-terminal signature / Proteasome alpha-type subunits signature. / Proteasome alpha-subunit, N-terminal domain / Proteasome subunit A N-terminal signature Add an annotation / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome B-type subunit / Proteasome beta-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal
Similarity search - Domain/homology
Proteasome subunit alpha / Proteasome subunit beta
Similarity search - Component
Biological speciesThermoplasma acidophilum (acidophilic)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsHerzik Jr MA / Wu M / Lander GC
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering (NIH/NIBIB)DP2EB020402 United States
CitationJournal: Nat Methods / Year: 2017
Title: Achieving better-than-3-Å resolution by single-particle cryo-EM at 200 keV.
Authors: Mark A Herzik / Mengyu Wu / Gabriel C Lander /
Abstract: Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible ...Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.
History
DepositionMay 24, 2017-
Header (metadata) releaseJun 14, 2017-
Map releaseJun 14, 2017-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5vy4
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8742.png

Downloads & links

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Map

FileDownload / File: emd_8742.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal sharpened map of T. acidophilum 20S proteasome collected using image shift navigation
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 256 pix.
= 232.96 Å		0.91 Å/pix.
x 256 pix.
= 232.96 Å		0.91 Å/pix.
x 256 pix.
= 232.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.19424753 - 0.3151097
Average (Standard dev.)-0.00020631733 (±0.018873818)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 232.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.910.910.91
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z232.960232.960232.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1940.315-0.000

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Supplemental data

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Additional map: Unsharpened map of T. acidophilum 20S proteasome collected...

Fileemd_8742_additional.map
AnnotationUnsharpened map of T. acidophilum 20S proteasome collected using image shift navigation
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Thermoplasma acidophilum 20S Proteasome, even half map

Fileemd_8742_half_map_1.map
AnnotationThermoplasma acidophilum 20S Proteasome, even half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Thermoplasma acidophilum 20S Proteasome, odd half map

Fileemd_8742_half_map_2.map
AnnotationThermoplasma acidophilum 20S Proteasome, odd half 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
  • Complex: Thermoplasma acidophilum 20S proteasome
    • Protein or peptide: Proteasome subunit alpha
    • Protein or peptide: Proteasome subunit beta

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Supramolecule #1: Thermoplasma acidophilum 20S proteasome

SupramoleculeName: Thermoplasma acidophilum 20S proteasome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Thermoplasma acidophilum 20S proteasome purified from Escherichia coli
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 700 KDa

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Macromolecule #1: Proteasome subunit alpha

MacromoleculeName: Proteasome subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 24.776281 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: RAITVFSPDG RLFQVEYARE AVKKGSTALG MKFANGVLLI SDKKVRSRLI EQNSIEKIQL IDDYVAAVTS GLVADARVLV DFARISAQQ EKVTYGSLVN IENLVKRVAD QMQQYTQYGG VRPYGVSLIF AGIDQIGPRL FDCDPAGTIN EYKATAIGSG K DAVVSFLE ...String:
RAITVFSPDG RLFQVEYARE AVKKGSTALG MKFANGVLLI SDKKVRSRLI EQNSIEKIQL IDDYVAAVTS GLVADARVLV DFARISAQQ EKVTYGSLVN IENLVKRVAD QMQQYTQYGG VRPYGVSLIF AGIDQIGPRL FDCDPAGTIN EYKATAIGSG K DAVVSFLE REYKENLPEK EAVTLGIKAL KSSLEEGEEL KAPEIASITV GNKYRIYDQE EVKKFL

UniProtKB: Proteasome subunit alpha

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Macromolecule #2: Proteasome subunit beta

MacromoleculeName: Proteasome subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 14 / Enantiomer: LEVO / EC number: proteasome endopeptidase complex
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Molecular weightTheoretical: 22.294848 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TTTVGITLKD AVIMATERRV TMENFIMHKN GKKLFQIDTY TGMTIAGLVG DAQVLVRYMK AELELYRLQR RVNMPIEAVA TLLSNMLNQ VKYMPYMVQL LVGGIDTAPH VFSIDAAGGS VEDIYASTGS GSPFVYGVLE SQYSEKMTVD EGVDLVIRAI S AAKQRDSA ...String:
TTTVGITLKD AVIMATERRV TMENFIMHKN GKKLFQIDTY TGMTIAGLVG DAQVLVRYMK AELELYRLQR RVNMPIEAVA TLLSNMLNQ VKYMPYMVQL LVGGIDTAPH VFSIDAAGGS VEDIYASTGS GSPFVYGVLE SQYSEKMTVD EGVDLVIRAI S AAKQRDSA SGGMIDVAVI TRKDGYVQLP TDQIESRIRK LGLIL

UniProtKB: Proteasome subunit beta

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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: 7.4
Component:
ConcentrationFormulaName
25.0 mMC4H11NO3Tris
120.0 mMNaClsodium chloride
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec. / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.009000000000000001 kPa / Details: 15 Watts
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: 3 uL of sample/grid was manually blotted for 4 seconds prior to immediate plunge-freezing in liquid nitrogen-cooled ethane..

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Digitization - Frames/image: 1-68 / Number grids imaged: 1 / Number real images: 394 / Average exposure time: 17.0 sec. / Average electron dose: 65.0 e/Å2
Details: Images were collected using image shift navigation to target exposure.
Electron beamAcceleration voltage: 200 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: 2.8000000000000003 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

DetailsMovies were collected in super-resolution mode and Fourier-binned by two prior to motion correction and dose weighting using MotionCor2 program.
Particle selectionNumber selected: 111184
Details: Template-based particle picking was performed using templates generated from reference-free 2D classification of an initial set of automated particle picks.
Startup modelType of model: EMDB MAP
EMDB ID:

6283
EMDB Unreleased entry


Details: Initial model was low-passed filtered to 60 Angstrom resolution
Final reconstructionApplied symmetry - Point group: D7 (2x7 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 96254
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsStarting model was generated by stripping PDB entry 1YAR of all ligands and alternate conformations, then refining into the EM density using imposed symmetry while adjusting weighting/scoring according to estimated map resolution. The top 10 generated models (ranked based on quality metrics) were real-space refined using Phenix software.
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 99 / Target criteria: Maximum Likelihood
Output model

PDB-5vy4:
Thermoplasma acidophilum 20S Proteasome using 200keV with image shift

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