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- EMDB-20359: MicroED structure of proteinase K from an uncoated, single lamell... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-20359 | |||||||||
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Title | MicroED structure of proteinase K from an uncoated, single lamella at 2.17A resolution (#8) | |||||||||
![]() | MicroED map of proteinase K from a single uncoated lamella at 2.17A resolution (#8) | |||||||||
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Function / homology | ![]() peptidase K / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() | |||||||||
Method | electron crystallography / cryo EM / Resolution: 2.17 Å | |||||||||
![]() | Martynowycz MW / Zhao W / Hattne J / Jensen GJ / 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 | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 29.4 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 17.2 KB 17.2 KB | Display Display | ![]() |
Images | ![]() | 94.9 KB | ||
Filedesc structureFactors | ![]() | 356.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6pkmMC ![]() 6pkjC ![]() 6pkkC ![]() 6pklC ![]() 6pknC ![]() 6pkoC ![]() 6pkpC ![]() 6pkqC ![]() 6pkrC ![]() 6pksC ![]() 6pktC C: citing same article ( M: atomic model generated by this map |
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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
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | MicroED map of proteinase K from a single uncoated lamella at 2.17A resolution (#8) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X: 0.35146 Å / Y: 0.35146 Å / Z: 0.33009 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 28.93 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: Engyodontium album, Tritirachium album |
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 |
-Macromolecule #2: water
Macromolecule | Name: water / type: ligand / ID: 2 / Number of copies: 42 / 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 | 20 mg/mL |
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Buffer | pH: 7.5 |
Grid | Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12.0 nm / Pretreatment - Type: GLOW DISCHARGE / Details: unspecified |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 273.0 K / Instrument: FEI VITROBOT MARK IV |
Details | Proteinase K purchased from Sigma. |
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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: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 28.0 µm / Number grids imaged: 1 / Number real images: 100 / Number diffraction images: 100 / Average exposure time: 3.0 sec. / Average electron dose: 0.03 e/Å2 Details: Continuous rotation with a rotation rate of 0.2 degrees per second and a readout every 3 seconds |
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: 2055.0 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |