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- EMDB-7490: 1.71 A MicroED structure of proteinase K at 0.86 e- / A^2 -

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Basic information

Entry
Database: EMDB / ID: EMD-7490
Title1.71 A MicroED structure of proteinase K at 0.86 e- / A^2
Map data
SampleProteinase K
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / metal ion binding
Proteinase K-like catalytic domain / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase domain profile. / 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 / Serine proteases, subtilase family, serine active site. ...Proteinase K-like catalytic domain / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase domain profile. / 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 / Serine proteases, subtilase family, serine active site. / Peptidase S8/S53 domain superfamily / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Subtilase family / Peptidase inhibitor I9 / Serine proteases, subtilase family, aspartic acid active site.
Proteinase K
Biological speciesEngyodontium album (fungus) / Engyodontium album, Tritirachium album
Methodelectron crystallography / cryo EM / Resolution: 1.71 Å
AuthorsHattne J / Shi D / Glynn C / Zee C-T / Gallagher-Jones M / Martynowycz MW / Rodriguez JA / Gonen T
CitationJournal: Structure / Year: 2018
Title: Analysis of Global and Site-Specific Radiation Damage in Cryo-EM.
Authors: Johan Hattne / Dan Shi / Calina Glynn / Chih-Te Zee / Marcus Gallagher-Jones / Michael W Martynowycz / Jose A Rodriguez / Tamir Gonen /
Abstract: Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron ...Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.
Validation ReportPDB-ID: 6cl7

SummaryFull reportAbout validation report
History
DepositionMar 2, 2018-
Header (metadata) releaseMay 2, 2018-
Map releaseMay 16, 2018-
UpdateMay 16, 2018-
Current statusMay 16, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.393
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.393
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6cl7
  • Surface level: 0.393
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_7490.map.gz / Format: CCP4 / Size: 3.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.57 Å/pix.
x 93 pix.
= 100.722 Å
0.56 Å/pix.
x 90 pix.
= 67.01 Å
0.56 Å/pix.
x 100 pix.
= 67.01 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.55842 Å / Y: 0.55842 Å / Z: 0.57229 Å
Density
Contour LevelBy EMDB: 0.393 / Movie #1: 0.393
Minimum - Maximum-0.391335 - 1.6839701
Average (Standard dev.)-0.008173919 (±0.21355428)
SymmetrySpace group: 96
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-8-80-48
Dimensions9010093
Spacing120120176
CellA: 67.0104 Å / B: 67.0104 Å / C: 100.7223 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.558416666666670.558416666666670.57228409090909
M x/y/z120120176
origin x/y/z0.0000.0000.000
length x/y/z67.01067.010100.722
α/β/γ90.00090.00090.000
start NX/NY/NZ-8-80-48
NX/NY/NZ9010093
MAP C/R/S213
start NC/NR/NS-80-8-48
NC/NR/NS1009093
D min/max/mean-0.3911.684-0.008

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Supplemental data

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Sample components

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Entire Proteinase K

EntireName: Proteinase K / Number of components: 2

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Component #1: cellular-component, Proteinase K

Cellular-componentName: Proteinase K / Recombinant expression: No
MassTheoretical: 28.888994 kDa
SourceSpecies: Engyodontium 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: Engyodontium album, Tritirachium album

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Experimental details

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Sample preparation

SpecimenSpecimen state: 3D array / Method: cryo EM
Crystal parametersSpace group: P43212 / A: 67.0104 Å / B: 67.0104 Å / C: 100.722 Å / α: 90 %deg; / β: 90 %deg; / γ: 90 %deg;
Sample solutionSpecimen conc.: 25 mg/mL / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 30 %

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
ImagingMicroscope: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 0.0357 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: DIFFRACTION
Specimen HolderModel: OTHER
CameraDetector: TVIPS TEMCAM-F416 (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 289 / Sampling size: 31.2 µm

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Image processing

ProcessingMethod: electron crystallography
3D reconstructionResolution: 1.71 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES

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Atomic model buiding

Modeling #1Refinement space: RECIPROCAL / Details: Electron scattering factors
Input PDB model: 5I9S
Chain ID: A

Overall bvalue: 6.613
Output model

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