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- EMDB-40351: The MicroED structure of proteinase K crystallized by suspended d... -

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Basic information

Entry
Database: EMDB / ID: EMD-40351
TitleThe MicroED structure of proteinase K crystallized by suspended drop crystallization
Map data
Sample
  • Organelle or cellular component: Proteinase K from Tritirachium album
    • Protein or peptide: Proteinase K
  • Ligand: SULFATE ION
  • Ligand: CALCIUM ION
  • Ligand: water
KeywordsMicroED / suspended drop crystallization / enzyme / 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 / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain
Similarity search - Domain/homology
Biological speciesParengyodontium album (fungus)
Methodelectron crystallography / cryo EM / Resolution: 2.1 Å
AuthorsGillman C / Nicolas WJ / Martynowycz MW / Gonen T
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
Department of Defense (DOD, United States)HDTRA1-21-1-0004 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: IUCrJ / Year: 2023
Title: Design and implementation of suspended drop crystallization.
Authors: Cody Gillman / William J Nicolas / Michael W Martynowycz / Tamir Gonen /
Abstract: In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an ...In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.
History
DepositionApr 6, 2023-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40351.png

Downloads & links

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Map

FileDownload / File: emd_40351.map.gz / Format: CCP4 / Size: 6.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.47 Å/pix.
x 111 pix.
= 52.617 Å		0.47 Å/pix.
x 126 pix.
= 59.728 Å		0.51 Å/pix.
x 117 pix.
= 59.641 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.47403 Å / Y: 0.47403 Å / Z: 0.50975 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.470045
Minimum - Maximum-3.7039838 - 5.5067077
Average (Standard dev.)0.000104513456 (±0.9800252)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-101-59-10
Dimensions126117111
Spacing111126117
CellA: 52.617085 Å / B: 59.7275 Å / C: 59.64075 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Proteinase K from Tritirachium album

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

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Supramolecule #1: Proteinase K from Tritirachium album

SupramoleculeName: Proteinase K from Tritirachium album / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Parengyodontium album (fungus)
Molecular weightTheoretical: 28.9 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: Parengyodontium album (fungus)
Molecular weightTheoretical: 28.930783 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 LDIFGPGTSI LSTWIGGSTR SISGTSMATP HVAGLAAYLM TL GKTTAAS ACRYIADTAN KGDLSNIPFG TVNLLAYNNY QA

UniProtKB: Proteinase K

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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

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 108 / 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

Concentration25 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 700 / Number diffraction images: 700 / Average electron dose: 0.64 e/Å2
Details: Detector distance: 1550 mm Images collected 60 degrees from -30 to +30 tilt
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Camera length: 1550 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Tilt angle: -30.0, 30.0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.1 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Crystallography statisticsNumber intensities measured: 56301 / Number structure factors: 12774 / Fourier space coverage: 87 / R merge: 44.5 / Overall phase residual: 0 / Phase error rejection criteria: none / High resolution: 2.1 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.1 Å / Shell - Low resolution: 30.42 Å / Shell - Number structure factors: 12774 / Shell - Phase residual: 31 / Shell - Fourier space coverage: 87 / Shell - Multiplicity: 4.41

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