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- EMDB-22463: Proteinase K soaked with I3C determined by MicroED from a single ... -

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Basic information

Entry
Database: EMDB / ID: EMD-22463
TitleProteinase K soaked with I3C determined by MicroED from a single milled microcrystal
Map data2Fo-Fc map
Sample
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid
  • 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)
Methodelectron crystallography / cryo EM / Resolution: 1.78 Å
AuthorsMartynowycz MW / Gonen T
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
CitationJournal: Structure / Year: 2021
Title: Ligand Incorporation into Protein Microcrystals for MicroED by On-Grid Soaking.
Authors: Michael W Martynowycz / Tamir Gonen /
Abstract: A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy ...A high throughout method for soaking ligands into protein microcrystals on TEM grids is presented. Every crystal on the grid is soaked simultaneously using only standard cryoelectron microscopy vitrification equipment. The method is demonstrated using proteinase K microcrystals soaked with the 5-amino-2,4,6-triodoisophthalic acid (I3C) magic triangle. A soaked microcrystal is milled to a thickness of approximately 200 nm using a focused ion beam, and MicroED data are collected. A high-resolution structure of the protein with four ligands at high occupancy is determined. Both the number of ligands bound and their occupancy is higher using on-grid soaking of microcrystals compared with much larger crystals treated similarly and investigated by X-ray crystallography. These results indicate that on-grid soaking ligands into microcrystals results in efficient uptake of ligands into protein microcrystals.
History
DepositionAug 16, 2020-
Header (metadata) releaseOct 14, 2020-
Map releaseOct 14, 2020-
UpdateJan 20, 2021-
Current statusJan 20, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jsy
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7jsy
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22463.png

Downloads & links

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Map

FileDownload / File: emd_22463.map.gz / Format: CCP4 / Size: 6.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2Fo-Fc map
Voxel sizeX=Y=Z: 0.56296 Å
Density
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-2.4631534 - 8.698446
Average (Standard dev.)0.0011024013 (±1.001575)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-94-60-19
Dimensions128118114
Spacing114128118
CellA: 64.17772 Å / B: 72.0592 Å / C: 66.42957 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.56296491228070.56296093750.56296610169492
M x/y/z114128118
origin x/y/z0.0000.0000.000
length x/y/z64.17872.05966.430
α/β/γ90.00090.00090.000
start NX/NY/NZ-19-94-60
NX/NY/NZ114128118
MAP C/R/S321
start NC/NR/NS-60-94-19
NC/NR/NS118128114
D min/max/mean-2.4638.6980.001

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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: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid
  • Ligand: water

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Supramolecule #1: Proteinase K

SupramoleculeName: Proteinase K / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Serine protease
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

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Macromolecule #2: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid

MacromoleculeName: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid / type: ligand / ID: 2 / Number of copies: 4 / Formula: I3C
Molecular weightTheoretical: 558.835 Da
Chemical component information

ChemComp-I3C:
5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 246 / 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

Concentration5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Nominal defocus min: 0.0 µm / Camera length: 1900 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: -30.0, 30.0
TemperatureMin: 70.0 K
DetailsTilt series: -30 to +30, 0.25 deg/sec, 1 sec readout
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 28.0 µm / Number grids imaged: 1 / Number real images: 240 / Number diffraction images: 240 / Average exposure time: 1.0 sec. / Average electron dose: 0.01 e/Å2

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Image processing

Crystal parametersUnit cell - A: 67.55 Å / Unit cell - B: 67.55 Å / Unit cell - C: 102.77 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Space group: 96
Crystallography statisticsNumber intensities measured: 109326 / Number structure factors: 10458 / Fourier space coverage: 94.52 / R sym: 0.13 / R merge: 0.26 / Overall phase error: 18 / Overall phase residual: 18 / Phase error rejection criteria: 0 / High resolution: 1.78 Å / Shell - Shell ID: 1 / Shell - High resolution: 43.32 Å / Shell - Low resolution: 1.78 Å / Shell - Number structure factors: 10458 / Shell - Phase residual: 18 / Shell - Fourier space coverage: 94.52 / Shell - Multiplicity: 4.9
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 1.78 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES

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Atomic model buiding 1

Initial modelPDB ID:

6cl7


Chain - Chain ID: A / Chain - Residue range: 1-279
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 13.51 / Target criteria: maximum liklihood
Output model

PDB-7jsy:
Proteinase K soaked with I3C determined by MicroED from a single milled microcrystal

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