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- EMDB-20476: MicroED structure of proteinase K recorded on CetaD -

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Basic information

Entry
Database: EMDB / ID: EMD-20476
TitleMicroED structure of proteinase K recorded on CetaD
Map dataProteinase K recorded on CetaD
Sample
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: CALCIUM IONCalcium
  • Ligand: water
KeywordsHydrolase
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: 2.7 Å
AuthorsHattne J / Martynowycz MW
CitationJournal: IUCrJ / Year: 2019
Title: MicroED with the Falcon III direct electron detector.
Authors: Johan Hattne / Michael W Martynowycz / Pawel A Penczek / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, ...Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.
History
DepositionJul 17, 2019-
Header (metadata) releaseAug 28, 2019-
Map releaseAug 28, 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: 0.18084
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.18084
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6pu5
  • Surface level: 0.18084
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20476.png

Downloads & links

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Map

FileDownload / File: emd_20476.map.gz / Format: CCP4 / Size: 868.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationProteinase K recorded on CetaD
Voxel sizeX: 0.87726 Å / Y: 0.87726 Å / Z: 0.89714 Å
Density
Contour LevelBy AUTHOR: 0.18084 / Movie #1: 0.18084
Minimum - Maximum-0.4088772 - 0.5921484
Average (Standard dev.)-0.00032427532 (±0.12056238)
SymmetrySpace group: 96
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-13-52-30
Dimensions576659
Spacing7676112
CellA: 66.6714 Å / B: 66.6714 Å / C: 100.480194 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.877250.877250.89714285714286
M x/y/z7676112
origin x/y/z0.0000.0000.000
length x/y/z66.67166.671100.480
α/β/γ90.00090.00090.000
start NX/NY/NZ-13-52-30
NX/NY/NZ576659
MAP C/R/S213
start NC/NR/NS-52-13-30
NC/NR/NS665759
D min/max/mean-0.4090.592-0.000

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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: CALCIUM IONCalcium
  • Ligand: water

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Supramolecule #1: Proteinase K

SupramoleculeName: Proteinase K / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Parengyodontium album (fungus)
Molecular weightTheoretical: 28.96915 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: 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: water

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

Concentration20 mg/mL
BufferpH: 8 / Component - Concentration: 50.0 mM / Component - Formula: C4H11NO3 / Component - Name: Tris
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 2131 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 100.0 K
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 165 / Number diffraction images: 165 / Average exposure time: 2.4069 sec. / Average electron dose: 0.024069 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Crystallography statisticsNumber intensities measured: 28788 / Number structure factors: 6520 / Fourier space coverage: 98 / R sym: 0.44 / R merge: 0.44 / Overall phase error: 43.59 / Overall phase residual: 43.59 / Phase error rejection criteria: 0 / High resolution: 2.7 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.7 Å / Shell - Low resolution: 2.83 Å / Shell - Number structure factors: 825 / Shell - Phase residual: 61.97 / Shell - Fourier space coverage: 97.2 / Shell - Multiplicity: 3.8
Molecular replacementSoftware - Name: MOLREP (ver. 11.7.01)
Symmetry determination software listSoftware - Name: POINTLESS (ver. 1.11.19)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: REFMAC (ver. 5.8.0238)
Merging software listSoftware - Name: AIMLESS (ver. 0.7.4)

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Atomic model buiding 1

Initial modelPDB ID:

5k7s


Chain - Chain ID: A / Chain - Residue range: 106-384 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsElectron scattering factors
RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 23.033
Output model

PDB-6pu5:
MicroED structure of proteinase K recorded on CetaD

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