|Entry||Database: EMDB / ID: 0343|
|Title||MicroED structure of Proteinase K at 2.75A resolution from a single milled crystal.|
|Map data||MicroED 2Fo-Fc map of proteinase K at 2.75A resolution from a single milled crystal.|
(ligand) x 3
|Function / homology||Proteinase K-like catalytic domain / Serine proteases, subtilase family, aspartic acid active site. / Serine proteases, subtilase family, serine active site. / Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase S8, subtilisin-related / Peptidase S8, subtilisin, His-active site / Peptidase S8, subtilisin, Asp-active site / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase family, histidine active site. ...Proteinase K-like catalytic domain / Serine proteases, subtilase family, aspartic acid active site. / Serine proteases, subtilase family, serine active site. / Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase S8, subtilisin-related / Peptidase S8, subtilisin, His-active site / Peptidase S8, subtilisin, Asp-active site / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase family, histidine active site. / Peptidase S8/S53 domain superfamily / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Subtilase family / Peptidase inhibitor I9 / peptidase K / serine-type endopeptidase activity / metal ion binding / Proteinase K|
Function and homology information
|Source||Engyodontium album (fungus)|
|Method||electron crystallography / cryo EM / 2.75 Å resolution|
|Authors||Martynowycz MW / Zhao W / Hattne J / Jensen GJ / Gonen T|
|Citation||Journal: Structure / Year: 2018|
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.
|Validation Report||PDB-ID: 6n4u|
SummaryFull reportAbout validation report
|Date||Deposition: Nov 20, 2018 / Header (metadata) release: Feb 6, 2019 / Map release: Feb 6, 2019 / Last update: Feb 6, 2019|
|Structure viewer||EM map: |
Downloads & links
|File||emd_0343.map.gz (map file in CCP4 format, 1707 KB)|
|Projections & slices|
Images are generated by Spider.
(generated in cubic-lattice coordinate)
|Voxel size||X: 0.6746 Å / Y: 0.6746 Å / Z: 0.66669 Å|
CCP4 map header:
+Entire Proteinase K
|Entire||Name: Proteinase K / Number of components: 5|
+Component #1: cellular-component, Proteinase K
|Cellular-component||Name: Proteinase K / Recombinant expression: No|
|Mass||Theoretical: 28.93 kDa|
|Source||Species: Engyodontium album (fungus)|
+Component #2: protein, Proteinase K
|Protein||Name: Proteinase K / Number of Copies: 1 / Recombinant expression: No|
|Mass||Theoretical: 28.958791 kDa|
|Source||Species: Engyodontium album (fungus)|
|Source (engineered)||Expression System: Engyodontium album (fungus)|
+Component #3: ligand, CALCIUM ION
|Ligand||Name: CALCIUM IONCalcium / Number of Copies: 2 / Recombinant expression: No|
|Mass||Theoretical: 4.007805 MDa|
+Component #4: ligand, SULFATE ION
|Ligand||Name: SULFATE IONSulfate / Number of Copies: 1 / Recombinant expression: No|
|Mass||Theoretical: 9.606305 MDa|
+Component #5: ligand, water
|Ligand||Name: water / Number of Copies: 23 / Recombinant expression: No|
|Mass||Theoretical: 1.801505 MDa|
|Specimen||Specimen state: 3D array / Method: cryo EM|
|Crystal parameters||Space group: P43 21 2 / A: 67.46 Å / B: 67.46 Å / C: 106.67 Å / Alpha: 90 deg. / Beta: 90 deg. / Gamma: 90 deg.|
|Sample solution||Specimen conc.: 20 mg/ml / pH: 7.5|
|Vitrification||Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 273 K / Humidity: 100 %|
-Electron microscopy imaging
Model: Talos Arctica / Image courtesy: FEI Company
|Imaging||Microscope: FEI TALOS ARCTICA|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 0.04 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: DIFFRACTION|
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 77.0 - 90.0 K)|
|Camera||Detector: FEI CETA (4k x 4k)|
|Image acquisition||Sampling size: 28 microns|
Details: Continuous rotation from -30 to +30 with a rotation rate of 0.2 degrees per second and a readout every 3 seconds.
|Processing||Method: electron crystallography / Details: This was the new CetaD.|
|3D reconstruction||CTF correction: NONE / Resolution: 2.75 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES|
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