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- EMDB-0343: MicroED structure of Proteinase K at 2.75A resolution from a sing... -

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Basic information

Entry
Database: EMDB / ID: EMD-0343
TitleMicroED structure of Proteinase K at 2.75A resolution from a single milled crystal.
Map dataMicroED 2Fo-Fc map of proteinase K at 2.75A resolution from a single milled crystal.
Sample
  • Organelle or cellular component: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: CALCIUM ION
  • Ligand: SULFATE ION
  • Ligand: water
Keywordsbroad-spectrum serum proteinase / HYDROLASE
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / proteolysis / extracellular space / 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. ...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 speciesEngyodontium album (fungus)
Methodelectron crystallography / cryo EM / Resolution: 2.75 Å
AuthorsMartynowycz MW / Zhao W
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R35 GM122588 United States
CitationJournal: Structure / Year: 2019
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
DepositionNov 20, 2018-
Header (metadata) releaseFeb 6, 2019-
Map releaseFeb 6, 2019-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.46937
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.46937
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6n4u
  • Surface level: 1.46937
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0343.png

Downloads & links

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Map

FileDownload / File: emd_0343.map.gz / Format: CCP4 / Size: 1.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicroED 2Fo-Fc map of proteinase K at 2.75A resolution from a single milled crystal.
Voxel sizeX: 0.6746 Å / Y: 0.6746 Å / Z: 0.66669 Å
Density
Contour LevelBy AUTHOR: 1.46937 / Movie #1: 1.46937
Minimum - Maximum-3.9357493 - 5.0805335
Average (Standard dev.)0.00043542887 (±0.97957665)
SymmetrySpace group: 96
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-63-38-16
Dimensions797572
Spacing100100160
CellA: 67.46 Å / B: 67.46 Å / C: 106.67 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.67460.67460.6666875
M x/y/z100100160
origin x/y/z0.0000.0000.000
length x/y/z67.46067.460106.670
α/β/γ90.00090.00090.000
start NX/NY/NZ-16-63-38
NX/NY/NZ727975
MAP C/R/S321
start NC/NR/NS-38-63-16
NC/NR/NS757972
D min/max/mean-3.9365.0810.000

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Supplemental data

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Sample components

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Entire : Proteinase K

EntireName: Proteinase K
Components
  • Organelle or cellular component: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: CALCIUM ION
  • Ligand: SULFATE ION
  • Ligand: water

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Supramolecule #1: Proteinase K

SupramoleculeName: Proteinase K / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Engyodontium album (fungus)
Molecular weightTheoretical: 28.93 KDa

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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: Engyodontium album (fungus)
Molecular weightTheoretical: 28.958791 KDa
Recombinant expressionOrganism: Engyodontium album (fungus)
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

UniProtKB: Proteinase K

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: SULFATE ION

MacromoleculeName: SULFATE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: SO4
Molecular weightTheoretical: 96.063 Da
Chemical component information

ChemComp-SO4:
SULFATE ION

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 23 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

Concentration20 mg/mL
BufferpH: 7.5
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 273.0 K / Instrument: FEI VITROBOT MARK IV
DetailsProteinase K purchased from Sigma.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 77.0 K / Max: 90.0 K
Image recordingFilm 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 beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1700 mm
Sample stageSpecimen 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

DetailsThis was the new CetaD.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.75 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Molecular replacementSoftware - Name: PHENIX (ver. 2.8.2)
Crystallography statisticsNumber 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

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Atomic model buiding 1

Initial modelPDB ID:

5i9s


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 21.1 / Target criteria: R
Output model

PDB-6n4u:
MicroED structure of Proteinase K at 2.75A resolution from a single milled crystal.

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