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- EMDB-25464: MicroED structure of proteinase K from a 260 nm thick lamella mea... -
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Basic information
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Title | MicroED structure of proteinase K from a 260 nm thick lamella measured at 200 kV | |||||||||
![]() | 2Fo-Fc MicroED map from a 260 nm thick lamella measured at 200 kV | |||||||||
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![]() | Serine protease / HYDROLASE | |||||||||
Function / homology | ![]() peptidase K / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | electron crystallography / cryo EM | |||||||||
![]() | Martynowycz MW / Clabbers MTB | |||||||||
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
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 4.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14 KB 14 KB | Display Display | ![]() |
Images | ![]() | 100.5 KB | ||
Filedesc metadata | ![]() | 5.6 KB | ||
Filedesc structureFactors | ![]() | 328.2 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7sw6MC ![]() 7svyC ![]() 7svzC ![]() 7sw0C ![]() 7sw1C ![]() 7sw2C ![]() 7sw3C ![]() 7sw4C ![]() 7sw5C ![]() 7sw7C ![]() 7sw8C ![]() 7sw9C ![]() 7swaC ![]() 7swbC ![]() 7swcC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | 2Fo-Fc MicroED map from a 260 nm thick lamella measured at 200 kV | ||||||||||||||||||||
Voxel size | X: 0.4716 Å / Y: 0.4716 Å / Z: 0.47463 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 96 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
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Sample components
-Entire : Proteinase K
Entire | Name: Proteinase K |
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Components |
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-Supramolecule #1: Proteinase K
Supramolecule | Name: Proteinase K / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Serine protease |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 28.9 KDa |
-Macromolecule #1: Proteinase K
Macromolecule | Name: Proteinase K / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: peptidase K |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 28.930783 KDa |
Sequence | String: AAQTNAPWGL ARISSTSPGT STYYYDESAG QGSCVYVIDT GIEASHPEFE GRAQMVKTYY YSSRDGNGHG THCAGTVGSR TYGVAKKTQ LFGVKVLDDN GSGQYSTIIA GMDFVASDKN NRNCPKGVVA SLSLGGGYSS SVNSAAARLQ SSGVMVAVAA G NNNADARN ...String: AAQTNAPWGL ARISSTSPGT STYYYDESAG QGSCVYVIDT GIEASHPEFE GRAQMVKTYY YSSRDGNGHG THCAGTVGSR TYGVAKKTQ LFGVKVLDDN GSGQYSTIIA GMDFVASDKN NRNCPKGVVA SLSLGGGYSS SVNSAAARLQ SSGVMVAVAA G NNNADARN YSPASEPSVC TVGASDRYDR RSSFSNYGSV LDIFGPGTSI LSTWIGGSTR SISGTSMATP HVAGLAAYLM TL GKTTAAS ACRYIADTAN KGDLSNIPFG TVNLLAYNNY QA UniProtKB: Proteinase K |
-Macromolecule #2: water
Macromolecule | Name: water / type: ligand / ID: 2 / Number of copies: 127 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ![]() ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | electron crystallography |
Aggregation state | 3D array |
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Sample preparation
Concentration | 5 mg/mL |
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Buffer | pH: 7.5 |
Grid | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER |
Details | Microcrystals |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Temperature | Min: 77.0 K / Max: 90.0 K |
Image recording | Film or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 120 / Average exposure time: 1.0 sec. / Average electron dose: 0.01 e/Å2 Details: 0.5 degrees per second, 1 second readout, 30 to -30 degrees. |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1863 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Image processing
Details | Binned by 2. |
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Final reconstruction | Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: PHENIX |
Merging software list | Software - Name: AIMLESS |
Crystallography statistics | Number intensities measured: 88107 / Number structure factors: 16540 / Fourier space coverage: 91 / R sym: 13 / R merge: 28 / Overall phase error: 22 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 1.95 Å / Shell - Shell ID: 1 / Shell - High resolution: 1.95 Å / Shell - Low resolution: 2.07 Å / Shell - Number structure factors: 2561 / Shell - Phase residual: 29 / Shell - Fourier space coverage: 92 / Shell - Multiplicity: 5 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: A / Chain - Residue range: 106-384 / Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 20 / Target criteria: Maximum likelihood |
Output model | ![]() PDB-7sw6: |