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

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Basic information

Entry
Database: PDB / ID: 5vy4
TitleThermoplasma acidophilum 20S Proteasome using 200keV with image shift
Components
  • Proteasome subunit alpha
  • Proteasome subunit beta
KeywordsHYDROLASE / Proteasome
Function / homology
Function and homology information


proteasome endopeptidase complex / proteasome core complex, beta-subunit complex / threonine-type endopeptidase activity / proteasome core complex, alpha-subunit complex / 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 / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / 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 ...Peptidase T1A, proteasome beta-subunit, archaeal / Proteasome alpha subunit, archaeal / Aminohydrolase, N-terminal nucleophile (Ntn) domain / Glutamine Phosphoribosylpyrophosphate, subunit 1, domain 1 / 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 B-type subunit / Proteasome beta-type subunit profile. / : / Proteasome alpha-type subunit / Proteasome alpha-type subunit profile. / Proteasome subunit / Proteasome, subunit alpha/beta / Nucleophile aminohydrolases, N-terminal / 4-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Proteasome subunit alpha / Proteasome subunit beta
Similarity search - Component
Biological speciesThermoplasma acidophilum (acidophilic)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsHerzik Jr., M.A. / Wu, M. / Lander, G.C.
Funding support United States, 1items
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 14, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2017Group: Author supporting evidence / Data collection / Category: em_software / pdbx_audit_support
Item: _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Oct 25, 2017Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.3Nov 15, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Jul 18, 2018Group: Data collection / Experimental preparation / Category: em_sample_support / Item: _em_sample_support.grid_type
Revision 1.5Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-8742
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Proteasome subunit alpha
B: Proteasome subunit beta
C: Proteasome subunit alpha
D: Proteasome subunit beta
E: Proteasome subunit alpha
F: Proteasome subunit beta
G: Proteasome subunit alpha
H: Proteasome subunit beta
I: Proteasome subunit alpha
J: Proteasome subunit beta
K: Proteasome subunit alpha
L: Proteasome subunit beta
M: Proteasome subunit alpha
N: Proteasome subunit beta
O: Proteasome subunit alpha
P: Proteasome subunit beta
Q: Proteasome subunit alpha
R: Proteasome subunit beta
S: Proteasome subunit alpha
T: Proteasome subunit beta
U: Proteasome subunit alpha
V: Proteasome subunit beta
W: Proteasome subunit alpha
X: Proteasome subunit beta
Y: Proteasome subunit alpha
Z: Proteasome subunit beta
0: Proteasome subunit alpha
1: Proteasome subunit beta


Theoretical massNumber of molelcules
Total (without water)658,99628
Polymers658,99628
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area95470 Å2
ΔGint-178 kcal/mol
Surface area210100 Å2
Number of models10

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Components

#1: Protein
Proteasome subunit alpha / 20S proteasome subunit A / 20S proteasome alpha subunit / Proteasome core protein PsmA


Mass: 24776.281 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoplasma acidophilum (acidophilic) / Gene: psmA, Ta1288 / Production host: Escherichia coli (E. coli)
References: UniProt: P25156, proteasome endopeptidase complex
#2: Protein
Proteasome subunit beta / 20S proteasome subunit B / 20S proteasome beta subunit / Proteasome core protein PsmB


Mass: 22294.848 Da / Num. of mol.: 14
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoplasma acidophilum (acidophilic) / Gene: psmB, Ta0612 / Production host: Escherichia coli (E. coli)
References: UniProt: P28061, proteasome endopeptidase complex

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Thermoplasma acidophilum 20S proteasome / Type: COMPLEX
Details: Thermoplasma acidophilum 20S proteasome purified from Escherichia coli
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.7 MDa / Experimental value: NO
Source (natural)Organism: Thermoplasma acidophilum (acidophilic)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTrisC4H11NO31
2120 mMsodium chlorideNaCl1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 Watts / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
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 imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 2800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 17 sec. / Electron dose: 65 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 394
Details: Images were collected using image shift navigation to target exposure.
Image scansSampling size: 5 µm / Width: 7420 / Height: 7676 / Movie frames/image: 68 / Used frames/image: 1-68

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2580: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1FindEMparticle selectionTemplate-based correlation
2Leginon3.2image acquisitionAutomatic data acquisition software
4CTFFIND4CTF correctionWhole-micrograph CTF estimation
5Gctf1.06CTF correctionPer-particle local defocus determination
6RELION1.4CTF correction
9Rosetta2016.32.5883model fitting
11PHENIX1.11.1-2580model refinement
12RELION2initial Euler assignment
13RELION2final Euler assignment
15RELION23D reconstruction
Image processingDetails: Movies were collected in super-resolution mode and Fourier-binned by two prior to motion correction and dose weighting using MotionCor2 program.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles 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.
SymmetryPoint symmetry: D7 (2x7 fold dihedral)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 96254 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 99 / Protocol: OTHER / Space: REAL / Target criteria: Maximum Likelihood
Details: Starting 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 ...Details: Starting 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.
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01246480
ELECTRON MICROSCOPYf_angle_d1.01462734
ELECTRON MICROSCOPYf_dihedral_angle_d8.27728462
ELECTRON MICROSCOPYf_chiral_restr0.0617350
ELECTRON MICROSCOPYf_plane_restr0.0068050

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