|Entry||Database: EMDB / ID: EMD-0865|
|Title||MicroED structure of proteinase K at 1.50A determained using crystal lamellas prepared by focused ion beam milling|
Proteinase K / (ligand) x 2
|Function / homology|
Function and homology information
peptidase K / serine-type endopeptidase activity / metal ion binding
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 / Proteinase K-like catalytic domain / Peptidase S8/S53 domain superfamily / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily
|Biological species||Parengyodontium album (fungus) / Tritirachium album,Engyodontium album|
|Method||electron crystallography / cryo EM / Resolution: 1.5 Å|
|Authors||Zhou H / Luo Z / Li X|
|Funding support|| China, 4 items |
|Citation||Journal: J. Struct. Biol. / Year: 2019|
Title: Using focus ion beam to prepare crystal lamella for electron diffraction.
Authors: Heng Zhou / Zhipu Luo / Xueming Li /
Abstract: Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the ...Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the electron crystallographic method on large protein crystals with micrometer or larger sizes. Here, we used the focused ion beam (FIB) equipped on the scanning electron microscope (SEM) to mill a large crystal to thin lamella. The influences of the milling on the crystal lamella were observed and investigated, including radiation damage on the crystal surface during the FIB imaging, deformation of the thin crystal lamella, and variation in the diffraction intensities under electron radiation. These observations provide important information to optimize the FIB milling, and hence is important to obtain high-quality crystal samples for routine structure determination of protein crystals using the electron cryo-microscope.
|Validation Report||PDB-ID: 6law|
SummaryFull reportAbout validation report
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_0865.map.gz / Format: CCP4 / Size: 5.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
generated in cubic-lattice coordinate
|Voxel size||X: 0.4951 Å / Y: 0.4951 Å / Z: 0.50056 Å|
|Symmetry||Space group: 96|
CCP4 map header:
-Entire proteinase K
|Entire||Name: proteinase K / Number of components: 4|
-Component #1: protein, proteinase K
|Protein||Name: proteinase K / Recombinant expression: No|
|Source||Species: Parengyodontium 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: Tritirachium album,Engyodontium album|
-Component #3: ligand, SULFATE ION
|Ligand||Name: SULFATE IONSulfate / Number of Copies: 1 / Recombinant expression: No|
|Mass||Theoretical: 9.606305 MDa|
-Component #4: ligand, water
|Ligand||Name: water / Number of Copies: 230 / Recombinant expression: No|
|Mass||Theoretical: 1.801505 MDa|
|Specimen||Specimen state: 3D array / Method: cryo EM|
|Crystal parameters||Space group: P43212 / A: 69.31 Å / B: 69.31 Å / C: 104.12 Å / α: 90 %deg; / β: 90 %deg; / γ: 90 %deg;|
|Sample solution||pH: 7.5|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 0.028 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Imaging mode: DIFFRACTION|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
|Processing||Method: electron crystallography|
|3D reconstruction||Resolution: 1.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES|
-Atomic model buiding
|Modeling #1||Refinement protocol: flexible / Refinement space: RECIPROCAL|
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