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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
Sample
  • Complex: proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: SULFATE IONSulfate
  • Ligand: water
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / proteolysis / extracellular region / metal ion binding
Similarity search - Function
Proteinase K-like catalytic domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site ...Proteinase K-like catalytic domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase domain profile. / Peptidase S8, subtilisin-related / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain
Similarity search - Domain/homology
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.
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
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  • Surface view colored by radius
  • Surface level: 0.165
  • Imaged by UCSF Chimera
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  • 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 modelsPDB-6law
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0865.png

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)
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
Components
  • Complex: proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: SULFATE IONSulfate
  • Ligand: water

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Supramolecule #1: proteinase K

SupramoleculeName: proteinase K / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Parengyodontium album (fungus)

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Macromolecule #1: Proteinase K

MacromoleculeName: Proteinase K / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: peptidase K
Source (natural)Organism: Tritirachium album,Engyodontium album
Molecular weightTheoretical: 28.958791 KDa
SequenceString: 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

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Macromolecule #2: SULFATE ION

MacromoleculeName: SULFATE ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: SO4
Molecular weightTheoretical: 96.063 Da
Chemical component information

ChemComp-SO4:
SULFATE ION / Sulfate

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 230 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state3D array

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1000 mm
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 0.028 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit cell - A: 69.31 Å / Unit cell - B: 69.31 Å / Unit cell - C: 104.12 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Space group: P 43 21 2
Crystallography statisticsNumber intensities measured: 200471 / Number structure factors: 37742 / Fourier space coverage: 91.1 / R sym: 0.245 / R merge: 0.245 / Overall phase error: 0 / Overall phase residual: 1 / Phase error rejection criteria: 60 / High resolution: 1.5 Å / Shell - Shell ID: 1 / Shell - High resolution: 1.5 Å / Shell - Low resolution: 1.55 Å / Shell - Number structure factors: 3693 / Shell - Phase residual: 1 / Shell - Fourier space coverage: 90.6 / Shell - Multiplicity: 5.2
Final reconstructionResolution.type: BY AUTHOR / Resolution: 1.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT
Output model

PDB-6law:
MicroED structure of proteinase K at 1.50A determained using crystal lamellas prepared by focused ion beam milling

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