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- EMDB-0343: MicroED structure of Proteinase K at 2.75A resolution from a sing... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-0343 | |||||||||
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Title | MicroED structure of Proteinase K at 2.75A resolution from a single milled crystal. | |||||||||
![]() | MicroED 2Fo-Fc map of proteinase K at 2.75A resolution from a single milled crystal. | |||||||||
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![]() | broad-spectrum serum proteinase / 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 / Resolution: 2.75 Å | |||||||||
![]() | Martynowycz MW / Zhao W | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size. Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen / ![]() Abstract: Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation ...Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for 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 | ![]() | 1.5 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 14.7 KB 14.7 KB | Display Display | ![]() |
Images | ![]() | 94.9 KB | ||
Filedesc metadata | ![]() | 6.2 KB | ||
Filedesc structureFactors | ![]() | 316.7 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6n4uMC 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
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | MicroED 2Fo-Fc map of proteinase K at 2.75A resolution from a single milled crystal. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X: 0.6746 Å / Y: 0.6746 Å / Z: 0.66669 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 96 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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: organelle_or_cellular_component / 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: ![]() |
Molecular weight | Theoretical: 28.958791 KDa |
Recombinant expression | Organism: ![]() |
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 LDIFGPGTDI LSTWIGGSTR SISGTSMATP HVAGLAAYLM TL GKTTAAS ACRYIADTAN KGDLSNIPFG TVNLLAYNNY QA UniProtKB: Proteinase K |
-Macromolecule #2: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Macromolecule #3: SULFATE ION
Macromolecule | Name: SULFATE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: SO4 |
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Molecular weight | Theoretical: 96.063 Da |
Chemical component information | ![]() ChemComp-SO4: |
-Macromolecule #4: water
Macromolecule | Name: water / type: ligand / ID: 4 / Number of copies: 23 / 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 | Model: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: OTHER |
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 / Number grids imaged: 1 / Number diffraction images: 100 / Average exposure time: 2.0 sec. / Average electron dose: 0.04 e/Å2 Details: Continuous rotation from -30 to +30 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: 1700 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 | This was the new CetaD. |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 2.75 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES |
Molecular replacement | Software - Name: PHENIX (ver. 2.8.2) |
Crystallography statistics | Number intensities measured: 31013 / Number structure factors: 10807 / Fourier space coverage: 88.4 / R sym: 0.39 / R merge: 0.47 / Overall phase error: 22.41 / Overall phase residual: 22.41 / Phase error rejection criteria: 0 / High resolution: 2.75 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.75 Å / Shell - Low resolution: 3.1483 Å / Shell - Number structure factors: 1831 / Shell - Phase residual: 28.11 / Shell - Fourier space coverage: 88.23 / Shell - Multiplicity: 2.9 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Refinement | Space: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 21.1 / Target criteria: R |
Output model | ![]() PDB-6n4u: |