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Yorodumi- PDB-8t1n: Micro-ED Structure of a Novel Domain of Unknown Function Solved w... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8t1n | ||||||
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Title | Micro-ED Structure of a Novel Domain of Unknown Function Solved with AlphaFold | ||||||
Components | DUF1842 domain-containing protein | ||||||
Keywords | UNKNOWN FUNCTION / AlphaFold / Bacterial Protein / Domain of Unknown Function / Novel Fold / Micro-ED | ||||||
Function / homology | Domain of unknown function DUF1842 / Domain of unknown function DUF1843 / Domain of unknown function (DUF1842) / Domain of unknown function (DUF1843) / DUF1842 domain-containing protein Function and homology information | ||||||
Biological species | Burkholderia pseudomallei (bacteria) | ||||||
Method | ELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 3 Å | ||||||
Authors | Miller, J.E. / Cascio, D. / Sawaya, M.R. / Cannon, K.A. / Rodriguez, J.A. / Yeates, T.O. | ||||||
Funding support | United States, 1items
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Citation | Journal: Acta Crystallogr D Struct Biol / Year: 2024 Title: AlphaFold-assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography. Authors: Justin E Miller / Matthew P Agdanowski / Joshua L Dolinsky / Michael R Sawaya / Duilio Cascio / Jose A Rodriguez / Todd O Yeates / Abstract: Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent ...Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8t1n.cif.gz | 98.9 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8t1n.ent.gz | 72 KB | Display | PDB format |
PDBx/mmJSON format | 8t1n.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8t1n_validation.pdf.gz | 406.3 KB | Display | wwPDB validaton report |
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Full document | 8t1n_full_validation.pdf.gz | 410 KB | Display | |
Data in XML | 8t1n_validation.xml.gz | 10.5 KB | Display | |
Data in CIF | 8t1n_validation.cif.gz | 13.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/t1/8t1n ftp://data.pdbj.org/pub/pdb/validation_reports/t1/8t1n | HTTPS FTP |
-Related structure data
Related structure data | 8t0bSC 8t1mC S: Starting model for refinement C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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Unit cell |
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-Components
#1: Protein | Mass: 23314.254 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Burkholderia pseudomallei (bacteria) / Gene: BPSS0212 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q63NT7 |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON CRYSTALLOGRAPHY |
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EM experiment | Aggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography |
-Sample preparation
Component | Name: DUF1842 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Burkholderia pseudomallei (bacteria) | ||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) | ||||||||||||||||||||
Buffer solution | pH: 5.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Data collection
Experimental equipment | Model: Tecnai F30 / Image courtesy: FEI Company | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Microscopy | Model: FEI TECNAI F30 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Electron lens | Mode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Specimen holder | Cryogen: NITROGEN | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Image recording | Average exposure time: 10 sec. / Electron dose: 2 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of diffraction images: 81 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Image scans | Width: 2048 / Height: 2048 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
EM diffraction shell | Resolution: 3→47.25 Å / Fourier space coverage: 58.8 % / Multiplicity: 8.23 / Num. of structure factors: 4846 / Phase residual: 0.1 ° | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
EM diffraction stats | Fourier space coverage: 58.8 % / High resolution: 3 Å / Num. of intensities measured: 39900 / Num. of structure factors: 4846 / Phase error rejection criteria: not applicable / Rmerge: 36.5 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Detector | Date: Nov 1, 2019 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reflection | Highest resolution: 3.02 Å / Num. obs: 4846 / % possible obs: 58.8 % / CC1/2: 0.912 / Rmerge(I) obs: 0.365 / Rrim(I) all: 0.386 / Net I/σ(I): 5.58 / Num. measured all: 39900 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Reflection shell | Diffraction-ID: 1
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-Processing
EM 3D crystal entity | ∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 40.58 Å / B: 94.99 Å / C: 101.53 Å / Space group name: P212121 / Space group num: 19 | ||||||||||||||||||||||||||||||||||||||||
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: RECIPROCAL | ||||||||||||||||||||||||||||||||||||||||
Refinement | Method to determine structure: MOLECULAR REPLACEMENT Starting model: 8T0B Resolution: 3→3 Å / SU ML: 0.47 / Cross valid method: THROUGHOUT / σ(F): 1.39 / Phase error: 24.2 / Stereochemistry target values: ML
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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LS refinement shell |
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Refinement TLS params. | Method: refined / Origin x: 4.4667 Å / Origin y: 28.0391 Å / Origin z: -19.6696 Å
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Refinement TLS group | Selection details: all |