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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 data
SampleProteinase 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)
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: 2020
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.
Validation ReportPDB-ID: 7jsy

SummaryFull reportAbout validation report
History
DepositionAug 16, 2020-
Header (metadata) releaseOct 14, 2020-
Map releaseOct 14, 2020-
UpdateOct 14, 2020-
Current statusOct 14, 2020Processing 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

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)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.56 Å/pix.
x 114 pix.
= 64.178 Å
0.56 Å/pix.
x 128 pix.
= 72.059 Å
0.56 Å/pix.
x 118 pix.
= 66.43 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.56296 Å
Density
Contour LevelBy AUTHOR: 1 / 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 / Details: Serine protease / Number of components: 4

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Component #1: protein, Proteinase K

ProteinName: Proteinase K / Details: Serine protease / Recombinant expression: No
MassTheoretical: 28.9 kDa
SourceSpecies: Parengyodontium album (fungus)

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Component #2: protein, Proteinase K

ProteinName: Proteinase K / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 28.930783 kDa
SourceSpecies: Parengyodontium album (fungus)

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

LigandName: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 0.558835 kDa

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Component #4: ligand, water

LigandName: water / Number of Copies: 246 / 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: 96 / A: 67.55 Å / B: 67.55 Å / C: 102.77 Å / α: 90 %deg; / β: 90 %deg; / γ: 90 %deg;
Sample solutionSpecimen conc.: 5 mg/mL / pH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

ImagingMicroscope: TFS TALOS
Details: Tilt series: -30 to +30, 0.25 deg/sec, 1 sec readout
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 0.01 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: DIFFRACTION / Defocus: 0.0 - nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: (70.0 - K)
CameraDetector: FEI CETA (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 240 / Sampling size: 28 µm

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Image processing

ProcessingMethod: electron crystallography
3D reconstructionAlgorithm: FOURIER SPACE / Resolution: 1.78 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: maximum liklihood / Refinement space: RECIPROCAL
Input PDB model: 6CL7
Chain ID: A

Overall bvalue: 13.51
Output model

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