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- PDB-7skx: Ab initio structure of proteinase K from electron-counted MicroED data -
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Open data
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Basic information
Entry | Database: PDB / ID: 7skx | |||||||||
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Title | Ab initio structure of proteinase K from electron-counted MicroED data | |||||||||
![]() | Proteinase K | |||||||||
![]() | HYDROLASE | |||||||||
Function / homology | ![]() peptidase K / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | ELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.5 Å | |||||||||
![]() | Martynowycz, M.W. / Clabbers, M.T.B. / Hattne, J. / Gonen, T. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Ab initio phasing macromolecular structures using electron-counted MicroED data. Authors: Michael W Martynowycz / Max T B Clabbers / Johan Hattne / Tamir Gonen / ![]() Abstract: Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals ...Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data. | |||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 197.5 KB | Display | ![]() |
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PDB format | ![]() | 155.2 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 462.7 KB | Display | ![]() |
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Full document | ![]() | 463.3 KB | Display | |
Data in XML | ![]() | 8 KB | Display | |
Data in CIF | ![]() | 13.7 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 25185MC ![]() 7skwC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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1 |
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Unit cell |
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Components on special symmetry positions |
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Components
#1: Protein | Mass: 28930.783 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) ![]() | ||||||
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#2: Chemical | #3: Chemical | ChemComp-CA / #4: Water | ChemComp-HOH / | Has ligand of interest | N | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON CRYSTALLOGRAPHY |
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EM experiment | Aggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography |
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Sample preparation
Component | Name: Proteinase K / Type: COMPLEX / Entity ID: #1 / Source: NATURAL |
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Molecular weight | Value: 0.028 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() |
Buffer solution | pH: 7.5 |
Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Milled microcrystals |
Specimen support | Details: NEGATIVE / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2 |
Vitrification | Instrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K |
-Data collection
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: DIFFRACTION / Cs: 2.7 mm / C2 aperture diameter: 50 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: 0.5 sec. / Electron dose: 0.001 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 840 / Num. of grids imaged: 1 / Num. of real images: 1 Details: 0.15 degrees per second, 0.5 second readout, 30 to -30 degrees |
Image scans | Sampling size: 28 µm / Width: 2048 / Height: 2048 |
EM diffraction | Camera length: 1738 mm / Tilt angle list: -30,30 |
EM diffraction shell | Resolution: 1.53→1.5 Å / Fourier space coverage: 89.3 % / Multiplicity: 8.9 / Num. of structure factors: 1758 / Phase residual: 30 ° |
EM diffraction stats | Details: Phases were determined by placing 4 ideal helix fragments. These were extended by chain tracing and density modifications. Fourier space coverage: 98.87 % / High resolution: 1.5 Å / Num. of intensities measured: 416133 / Num. of structure factors: 39303 / Phase error: 20 ° / Phase error rejection criteria: None / Rmerge: 0.277 / Rsym: 0.087 |
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Processing
Software | Name: REFMAC / Version: 5.8.0267 / Classification: refinement / Contact author: Garib N. Murshudov / Contact author email: garib[at]mrc-lmb.cam.ac.uk / Date: 2020-24-08 Description: (un)restrained refinement or idealisation of macromolecular structures | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Binned by 2. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
EM 3D crystal entity | ∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.08 Å / B: 67.08 Å / C: 106.78 Å / Space group name: P43212 / Space group num: 96 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 14.137 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Maximum likelihood | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 1.5→43.386 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.95 / SU B: 4.529 / SU ML: 0.067 / Cross valid method: FREE R-VALUE / ESU R: 0.085 / ESU R Free: 0.078 Details: Hydrogens have been added in their riding positions
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Solvent computation | Ion probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 14.137 Å2
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Refinement step | Cycle: LAST / Resolution: 1.5→43.386 Å
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Refine LS restraints |
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