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- PDB-7sw0: MicroED structure of proteinase K from a 325 nm thick lamella mea... -
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Open data
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Basic information
Entry | Database: PDB / ID: 7sw0 | |||||||||
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Title | MicroED structure of proteinase K from a 325 nm thick lamella measured at 120 kV | |||||||||
![]() | Proteinase K | |||||||||
![]() | HYDROLASE / Serine protease | |||||||||
Function / homology | ![]() peptidase K / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | ELECTRON CRYSTALLOGRAPHY / electron crystallography / ![]() | |||||||||
![]() | Martynowycz, M.W. / Clabbers, M.T.B. / Unge, J. / Hattne, J. / Gonen, T. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Benchmarking the ideal sample thickness in cryo-EM. Authors: Michael W Martynowycz / Max T B Clabbers / Johan Unge / Johan Hattne / Tamir Gonen / ![]() Abstract: The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K ...The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods. | |||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 79.6 KB | Display | ![]() |
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PDB format | ![]() | 44.7 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 25458MC ![]() 7svyC ![]() 7svzC ![]() 7sw1C ![]() 7sw2C ![]() 7sw3C ![]() 7sw4C ![]() 7sw5C ![]() 7sw6C ![]() 7sw7C ![]() 7sw8C ![]() 7sw9C ![]() 7swaC ![]() 7swbC ![]() 7swcC ![]() 6cl7S |
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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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Unit cell |
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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: 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 / Details: Serine protease / Entity ID: #1 / Source: NATURAL |
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Molecular weight | Value: 0.0289 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: Microcrystals |
Specimen support | 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: 1 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 120 / Num. of grids imaged: 1 / Num. of real images: 1 Details: 0.5 degrees per second, 1 second readout, 30 to -30 degrees. |
Image scans | Sampling size: 28 µm / Width: 2048 / Height: 2048 |
EM diffraction | Camera length: 1427 mm |
EM diffraction shell | Resolution: 2.7→3.4 Å / Fourier space coverage: 83 % / Multiplicity: 6 / Num. of structure factors: 2499 / Phase residual: 33 ° |
EM diffraction stats | Fourier space coverage: 83 % / High resolution: 2.7 Å / Num. of intensities measured: 30326 / Num. of structure factors: 5453 / Phase error: 28 ° / Phase error rejection criteria: None / Rmerge: 64 / Rsym: 30 |
Detector | Date: Dec 11, 2020 |
Reflection | Resolution: 2.7→20 Å / Num. obs: 30326 / % possible obs: 82.6 % / Redundancy: 5.5 % / Biso Wilson estimate: 23.76 Å2 / CC1/2: 0.893 / Rmerge(I) obs: 0.64 / Rpim(I) all: 0.296 / Rrim(I) all: 0.708 / Net I/σ(I): 3.2 |
Reflection shell | Resolution: 2.7→2.86 Å / Rmerge(I) obs: 1.765 / Num. unique obs: 4720 / CC1/2: 0.359 / Rrim(I) all: 1.951 / Net I/σ(I) obs: 1.02 / % possible all: 83 |
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Processing
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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: 66.781 Å / B: 66.781 Å / C: 99.215 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: 15 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Maximum liklihood | ||||||||||||||||||||||||
Atomic model building | PDB-ID: 6CL7 Pdb chain-ID: A / Accession code: 6CL7 / Pdb chain residue range: 106-384 / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||
Refinement | Method to determine structure: ![]() Starting model: 6CL7 Resolution: 2.7→19.43 Å / SU ML: 0.3372 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.9284 Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 14.5 Å2 | ||||||||||||||||||||||||
Refinement step | Cycle: LAST / Resolution: 2.7→19.43 Å
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Refine LS restraints |
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LS refinement shell |
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