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- EMDB-0865: MicroED structure of proteinase K at 1.50A determained using crys... -

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Basic information

Entry
Database: EMDB / ID: EMD-0865
TitleMicroED structure of proteinase K at 1.50A determained using crystal lamellas prepared by focused ion beam milling
Map data
Sampleproteinase K:
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
Proteinase K
Biological speciesParengyodontium album (fungus) / Tritirachium album,Engyodontium album
Methodelectron crystallography / cryo EM / Resolution: 1.5 Å
AuthorsZhou H / Luo Z / Li X
Funding support China, 4 items
OrganizationGrant numberCountry
National Natural Science Foundation of China31570730 China
Ministry of Science and Technology (China)2016YFA0501102 China
National Natural Science Foundation of China31722015 China
Ministry of Science and Technology (China)2016YFA0501902 China
CitationJournal: 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 ReportPDB-ID: 6law

SummaryFull reportAbout validation report
History
DepositionNov 13, 2019-
Header (metadata) releaseDec 4, 2019-
Map releaseDec 4, 2019-
UpdateDec 4, 2019-
Current statusDec 4, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.165
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.165
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6law
  • Surface level: 0.165
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic models: PDB-6law
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0865.map.gz / Format: CCP4 / Size: 5.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.5 Å/pix.
x 111 pix.
= 104.116 Å
0.5 Å/pix.
x 111 pix.
= 69.314 Å
0.5 Å/pix.
x 123 pix.
= 69.314 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.4951 Å / Y: 0.4951 Å / Z: 0.50056 Å
Density
Contour LevelBy AUTHOR: 0.165 / Movie #1: 0.165
Minimum - Maximum-0.54019946 - 1.4676074
Average (Standard dev.)-0.00002671368 (±0.16420569)
SymmetrySpace group: 96
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin111-94150
Dimensions111123111
Spacing140140208
CellA: 69.313995 Å / B: 69.313995 Å / C: 104.11648 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.49510.49510.50055769230769
M x/y/z140140208
origin x/y/z0.0000.0000.000
length x/y/z69.31469.314104.116
α/β/γ90.00090.00090.000
start NX/NY/NZ111-94150
NX/NY/NZ111123111
MAP C/R/S213
start NC/NR/NS-94111150
NC/NR/NS123111111
D min/max/mean-0.5401.468-0.000

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Supplemental data

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Sample components

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Entire proteinase K

EntireName: proteinase K / Number of components: 4

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Component #1: protein, proteinase K

ProteinName: proteinase K / Recombinant expression: No
SourceSpecies: Parengyodontium album (fungus)

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Component #2: protein, Proteinase K

ProteinName: Proteinase K / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 28.958791 kDa
SourceSpecies: Tritirachium album,Engyodontium album

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Component #3: ligand, SULFATE ION

LigandName: SULFATE IONSulfate / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.606305 MDa

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Component #4: ligand, water

LigandName: water / Number of Copies: 230 / Recombinant expression: No
MassTheoretical: 1.801505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: 3D array / Method: cryo EM
Crystal parametersSpace group: P43212 / A: 69.31 Å / B: 69.31 Å / C: 104.12 Å / α: 90 %deg; / β: 90 %deg; / γ: 90 %deg;
Sample solutionpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 0.028 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: DIFFRACTION
Specimen HolderModel: OTHER
CameraDetector: GATAN ULTRASCAN 4000 (4k x 4k)

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Image processing

ProcessingMethod: electron crystallography
3D reconstructionResolution: 1.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: RECIPROCAL
Output model

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