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Yorodumi- PDB-6vrs: Single particle reconstruction of glucose isomerase from Streptom... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6vrs | ||||||||||||||||||
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Title | Single particle reconstruction of glucose isomerase from Streptomyces rubiginosus based on data acquired in the presence of substantial aberrations | ||||||||||||||||||
Components | xylose isomerase | ||||||||||||||||||
Keywords | ISOMERASE / glucose isomerase | ||||||||||||||||||
Function / homology | Function and homology information xylose isomerase / D-xylose metabolic process / xylose isomerase activity / magnesium ion binding / identical protein binding / cytoplasm Similarity search - Function | ||||||||||||||||||
Biological species | Streptomyces rubiginosus (bacteria) | ||||||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å | ||||||||||||||||||
Authors | Bromberg, R. / Guo, Y. / Borek, D. / Otwinowski, Z. | ||||||||||||||||||
Funding support | United States, 5items
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Citation | Journal: IUCrJ / Year: 2020 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: Journal: Biorxiv / Year: 2020 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 |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6vrs.cif.gz | 306.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6vrs.ent.gz | 246.6 KB | Display | PDB format |
PDBx/mmJSON format | 6vrs.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6vrs_validation.pdf.gz | 743.4 KB | Display | wwPDB validaton report |
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Full document | 6vrs_full_validation.pdf.gz | 749.6 KB | Display | |
Data in XML | 6vrs_validation.xml.gz | 42.2 KB | Display | |
Data in CIF | 6vrs_validation.cif.gz | 65.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/vr/6vrs ftp://data.pdbj.org/pub/pdb/validation_reports/vr/6vrs | HTTPS FTP |
-Related structure data
Related structure data | 21371MC 6vsaC 6vscC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10360 (Title: 2.7 Angstrom cryo-EM reconstructions of glucose isomerase in the presence of substantial aberrations Data size: 1.0 TB Data #1: Unaligned multiframe data for xylose isomerase (glucose isomerase) [micrographs - multiframe]) |
-Links
-Assembly
Deposited unit |
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments: Component-ID: 1 / Beg auth comp-ID: ASN / Beg label comp-ID: ASN / End auth comp-ID: GLY / End label comp-ID: GLY / Refine code: 1 / Auth seq-ID: 2 - 388 / Label seq-ID: 2 - 388
NCS ensembles :
NCS oper:
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-Components
#1: Protein | Mass: 43283.297 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Streptomyces rubiginosus (bacteria) / References: UniProt: P24300, xylose isomerase #2: Chemical | ChemComp-MN / #3: Water | ChemComp-HOH / | Has ligand of interest | Y | |
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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 |
-Sample preparation
Component | Name: glucose isomerase / Type: COMPLEX / Entity ID: #1 / Source: NATURAL |
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Molecular weight | Value: 0.1729 MDa / Experimental value: YES |
Source (natural) | Organism: Streptomyces rubiginosus (bacteria) |
Buffer solution | pH: 7.5 |
Buffer component | Conc.: 20 mM / Name: HEPES |
Specimen | Conc.: 40 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: GOLD / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-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: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
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: 5.3 mradians |
Image recording | Average exposure time: 100 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: 202 |
Image scans | Width: 3838 / Height: 3710 / Movie frames/image: 200 / Used frames/image: 1-200 |
-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: 114522 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: D2 (2x2 fold dihedral) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 61909 / Algorithm: BACK PROJECTION / Details: We used the approach implemented in cisTEM. / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: RECIPROCAL / Target criteria: REFMAC / Details: COOT was crucial as well. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5VR0 Accession code: 5VR0 / Source name: PDB / Type: experimental model | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 2.7→100.1 Å / Cor.coef. Fo:Fc: 0.972 / SU B: 11.452 / SU ML: 0.218 / ESU R: 0.417 Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
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Solvent computation | Solvent model: PARAMETERS FOR MASK CACLULATION | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 80.52 Å2
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Refinement step | Cycle: 1 / Total: 12208 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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