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- PDB-6vsa: Single particle reconstruction of HemQ from Geobacillus based on ... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6vsa | ||||||||||||||||||
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Title | Single particle reconstruction of HemQ from Geobacillus based on data acquired in the presence of substantial aberrations | ||||||||||||||||||
![]() | HemQ | ||||||||||||||||||
![]() | OXIDOREDUCTASE / heme-binding | ||||||||||||||||||
Function / homology | ![]() heme biosynthetic process / peroxidase activity / heme binding / metal ion binding Similarity search - Function | ||||||||||||||||||
Biological species | ![]() | ||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.32 Å | ||||||||||||||||||
![]() | Bromberg, R. / Guo, Y. / Borek, D. / Otwinowski, Z. | ||||||||||||||||||
Funding support | ![]()
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![]() | ![]() Title: High-resolution cryo-EM reconstructions in the presence of substantial aberrations. Authors: Raquel Bromberg / Yirui Guo / Dominika Borek / Zbyszek Otwinowski / ![]() Abstract: Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula ...Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula quantifying information loss owing to their presence is inferred that explains why Fourier-shell coefficient-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. The analysis is validated with reference-based aberration refinement for two cryo-EM SPR data sets acquired with a 200 kV microscope in the presence of coma exceeding 40 µm, and 2.3 and 2.7 Å reconstructions for 144 and 173 kDa particles, respectively, were obtained. The results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher order aberrations, and address inconsistent guidelines regarding the level of aberrations that is acceptable in cryo-EM SPR experiments. #1: ![]() Title: High-resolution cryo-EM reconstructions in the presence of substantial aberrations Authors: Bromberg, R. / Guo, Y. / Borek, D. / Otwinowski, Z. | ||||||||||||||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 248.2 KB | Display | ![]() |
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PDB format | ![]() | 198.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 21373MC ![]() 6vrsC ![]() 6vscC M: map data used to model this data C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 1.4 TB Data #1: Unaligned multiframe data for 2.3 Angstrom cryo-EM reconstructions of HemQ from Geobacillus in the presence of substantial aberrations [micrographs - multiframe]) |
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Links
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Assembly
Deposited unit | ![]()
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments: Component-ID: _ / Beg auth comp-ID: ALA / Beg label comp-ID: ALA / End auth comp-ID: VAL / End label comp-ID: VAL / Refine code: _ / Auth seq-ID: 4 - 248 / Label seq-ID: 4 - 248
NCS ensembles :
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Components
#1: Protein | Mass: 28817.609 Da / Num. of mol.: 5 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Strain: Y412MC52 / Gene: GYMC52_3505 / Plasmid: pMCSG7 / Production host: ![]() ![]() References: UniProt: A0A0E0TGF4, Oxidoreductases; Acting on a peroxide as acceptor; Peroxidases #2: Water | ChemComp-HOH / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: HemQ / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 0.144 MDa / Experimental value: YES |
Source (natural) | Organism: ![]() |
Source (recombinant) | Organism: ![]() ![]() |
Buffer solution | pH: 7.5 / Details: 100 mM NaCl |
Buffer component | Conc.: 20 mM / Name: HEPES |
Specimen | Conc.: 28 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
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Electron microscopy imaging
Microscopy | Model: TFS TALOS Details: The goal of the experiment was to show that it is possible to perform high resolution reconstruction in the presence of higher order aberrations. |
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Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC |
Specimen holder | Cryogen: NITROGEN / Residual tilt: 6.9 mradians |
Image recording | Average exposure time: 40 sec. / Electron dose: 120 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 268 / Details: 258 movies were used in the reconstruction. |
Image scans | Width: 3838 / Height: 3710 / Movie frames/image: 100 / Used frames/image: 1-100 |
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Processing
Software | Name: REFMAC / Version: 5.8.0257 / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 156210 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C5 (5 fold cyclic) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81302 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: RECIPROCAL / Target criteria: REFMAC / Details: COOT was crucial as well. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 1T0T Accession code: 1T0T / Source name: PDB / Type: experimental model | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 2.32→98.64 Å / Cor.coef. Fo:Fc: 0.979 / SU B: 7.012 / SU ML: 0.144 / ESU R: 0.196 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
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Solvent computation | Solvent model: PARAMETERS FOR MASK CACLULATION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 88.717 Å2
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Refine LS restraints |
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