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Yorodumi- PDB-6n4u: MicroED structure of Proteinase K at 2.75A resolution from a sing... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6n4u | ||||||
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Title | MicroED structure of Proteinase K at 2.75A resolution from a single milled crystal. | ||||||
Components | Proteinase K | ||||||
Keywords | HYDROLASE / broad-spectrum serum proteinase | ||||||
Function / homology | Function and homology information peptidase K / serine-type endopeptidase activity / proteolysis / extracellular region / metal ion binding Similarity search - Function | ||||||
Biological species | Engyodontium album (fungus) | ||||||
Method | ELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.75 Å | ||||||
Authors | Martynowycz, M.W. / Zhao, W. / Hattne, J. / Jensen, G.J. / Gonen, T. | ||||||
Funding support | United States, 1items
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Citation | Journal: Structure / Year: 2019 Title: Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size. Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen / Abstract: Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation ...Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED. | ||||||
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 | 6n4u.cif.gz | 65.2 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6n4u.ent.gz | 44.5 KB | Display | PDB format |
PDBx/mmJSON format | 6n4u.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/n4/6n4u ftp://data.pdbj.org/pub/pdb/validation_reports/n4/6n4u | HTTPS FTP |
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-Related structure data
-Links
-Assembly
Deposited unit |
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1 |
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Unit cell |
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-Components
#1: Protein | Mass: 28958.791 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Engyodontium album (fungus) / Gene: PROK / Production host: Engyodontium album (fungus) / References: UniProt: P06873, peptidase K | ||||
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#2: Chemical | #3: Chemical | ChemComp-SO4 / | #4: Water | ChemComp-HOH / | |
-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: Proteinase K / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL |
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Molecular weight | Value: 0.02893 MDa / Experimental value: NO |
Source (natural) | Organism: Engyodontium album (fungus) |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Proteinase K purchased from Sigma. |
Specimen support | Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 273 K |
-Data collection
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: DIFFRACTION / C2 aperture diameter: 100 µm / Alignment procedure: BASIC |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K |
Image recording | Average exposure time: 2 sec. / Electron dose: 0.04 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 100 / Num. of grids imaged: 1 Details: Continuous rotation from -30 to +30 with a rotation rate of 0.2 degrees per second and a readout every 3 seconds. |
Image scans | Sampling size: 28 µm / Width: 4096 / Height: 4096 |
EM diffraction | Camera length: 1700 mm |
EM diffraction shell | Resolution: 2.75→3.1483 Å / Fourier space coverage: 88.23 % / Multiplicity: 2.9 / Num. of structure factors: 1831 / Phase residual: 28.11 ° |
EM diffraction stats | Fourier space coverage: 88.4 % / High resolution: 2.75 Å / Num. of intensities measured: 31013 / Num. of structure factors: 10807 / Phase error: 22.41 ° / Phase residual: 22.41 ° / Phase error rejection criteria: 0 / Rmerge: 0.47 / Rsym: 0.39 |
-Processing
Software | Name: PHENIX / Version: (1.14_3260: ???) / Classification: refinement | ||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: This was the new CetaD. | ||||||||||||||||||||||||||||
EM 3D crystal entity | ∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.46 Å / B: 67.46 Å / C: 106.67 Å / Space group name: P43212 / Space group num: 96 | ||||||||||||||||||||||||||||
CTF correction | Details: NONE / Type: NONE | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.75 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL | ||||||||||||||||||||||||||||
Atomic model building | B value: 21.1 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: R | ||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 5I9S Accession code: 5I9S / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||||||
Refinement | Method to determine structure: MOLECULAR REPLACEMENT Starting model: 6CL7 Resolution: 2.75→43.546 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.25
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å | ||||||||||||||||||||||||||||
Refine LS restraints |
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LS refinement shell |
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