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- PDB-6pko: MicroED structure of proteinase K from a platinum coated, unpolis... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6pko | |||||||||
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Title | MicroED structure of proteinase K from a platinum coated, unpolished, single lamella at 2.07A resolution (#12) | |||||||||
![]() | 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: 2.07 Å | |||||||||
![]() | Martynowycz, M.W. / Zhao, W. / Hattne, J. / Jensen, G.J. / Gonen, T. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED. Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen / ![]() Abstract: Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction ...Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED. | |||||||||
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 | ![]() | 60.9 KB | Display | ![]() |
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PDB format | ![]() | 45.5 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 20361MC ![]() 6pkjC ![]() 6pkkC ![]() 6pklC ![]() 6pkmC ![]() 6pknC ![]() 6pkpC ![]() 6pkqC ![]() 6pkrC ![]() 6pksC ![]() 6pktC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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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
#1: Protein | Mass: 28930.783 Da / Num. of mol.: 1 / Fragment: UNP residues 106-384 / Source method: isolated from a natural source / Source: (natural) ![]() |
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#2: 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 |
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Sample preparation
Component | Name: Proteinase K / Type: COMPLEX / Entity ID: #1 / Source: NATURAL |
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Molecular weight | Value: 0.02893 MDa / Experimental value: NO |
Source (natural) | Organism: ![]() |
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 | Details: unspecified |
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: ![]() |
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: 3 sec. / Electron dose: 0.03 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 100 / Num. of grids imaged: 1 / Num. of real images: 100 Details: Continuous rotation 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: 2055 mm |
EM diffraction shell | Resolution: 2.07→33.66 Å / Fourier space coverage: 57.59 % / Multiplicity: 6.9 / Num. of structure factors: 8849 / Phase residual: 26.83 ° |
EM diffraction stats | Fourier space coverage: 57.59 % / High resolution: 2.07 Å / Num. of intensities measured: 60628 / Num. of structure factors: 8849 / Phase error: 26.83 ° / Phase residual: 26.83 ° / Phase error rejection criteria: 0 / Rmerge: 0.277 / Rsym: 0.29 |
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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: 67.3 Å / B: 67.3 Å / C: 105.1 Å / Space group name: 96 / Space group num: 96 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: NONE | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.07 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 29.2 / Protocol: OTHER / Space: RECIPROCAL | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6CL7 Pdb chain-ID: A / Pdb chain residue range: 106-384 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Refinement | Resolution: 2.07→33.66 Å / SU ML: 0.32 / Cross valid method: THROUGHOUT / σ(F): 1.35 / Phase error: 26.8
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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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