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- EMDB-25185: Ab initio structure of proteinase K from electron-counted MicroED data -
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Open data
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Basic information
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Title | Ab initio structure of proteinase K from electron-counted MicroED data | |||||||||
![]() | 1.5A resolution 2mFo-dFc MicroED map of proteinase K determined ab initio by electron-counted data | |||||||||
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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 | |||||||||
![]() | Martynowycz MW / Clabbers MTB / 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
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 9.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 15.8 KB 15.8 KB | Display Display | ![]() |
Images | ![]() | 49.3 KB | ||
Filedesc structureFactors | ![]() | 484.8 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7skxMC ![]() 7skwC 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 | 1.5A resolution 2mFo-dFc MicroED map of proteinase K determined ab initio by electron-counted data | ||||||||||||||||||||
Voxel size | X: 0.37267 Å / Y: 0.37267 Å / Z: 0.37076 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 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 28 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: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid
Macromolecule | Name: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid / type: ligand / ID: 2 / Number of copies: 2 / Formula: I3C |
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Molecular weight | Theoretical: 558.835 Da |
Chemical component information | ![]() ChemComp-I3C: |
-Macromolecule #3: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Macromolecule #4: water
Macromolecule | Name: water / type: ligand / ID: 4 / Number of copies: 234 / 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: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: NEGATIVE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER |
Details | Milled microcrystals |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 77.0 K / Max: 90.0 K |
Image recording | Film or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Sampling interval: 28.0 µm / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 840 / Average exposure time: 0.5 sec. / Average electron dose: 0.001 e/Å2 Details: 0.15 degrees per second, 0.5 second readout, 30 to -30 degrees |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Camera length: 1738 mm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: -30.0, 30.0 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Refinement | Space: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 14.137 / Target criteria: Maximum likelihood |
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Output model | ![]() PDB-7skx: |