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- EMDB-46871: Structure of proteinase K from energy-filtered MicroED data -

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Basic information

Entry
Database: EMDB / ID: EMD-46871
TitleStructure of proteinase K from energy-filtered MicroED data
Map dataMicroED 2mFo-DFc map of proteinase K
Sample
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: CALCIUM ION
  • Ligand: NITRATE ION
  • Ligand: water
Keywordsserine protease / 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: 1.09 Å
AuthorsClabbers MTB / Hattne J / Martynoqycz 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: bioRxiv / Year: 2024
Title: Energy filtering enables macromolecular MicroED data at sub-atomic resolution.
Authors: Max T B Clabbers / Johan Hattne / Michael W Martynowycz / Tamir Gonen /
Abstract: High resolution information is important for accurate structure modelling. However, this level of detail is typically difficult to attain in macromolecular crystallography because the diffracted ...High resolution information is important for accurate structure modelling. However, this level of detail is typically difficult to attain in macromolecular crystallography because the diffracted intensities rapidly fade with increasing resolution. The problem cannot be circumvented by increasing the fluence as this leads to detrimental radiation damage. Previously, we demonstrated that high quality MicroED data can be obtained at low flux conditions using electron counting with direct electron detectors. The improved sensitivity and accuracy of these detectors essentially eliminate the read-out noise, such that the measurement of faint high-resolution reflections is limited by other sources of noise. Inelastic scattering is a major contributor of such noise, increasing background counts and broadening diffraction spots. Here, we demonstrate that a substantial improvement in signal-to-noise ratio can be achieved using an energy filter to largely remove the inelastically scattered electrons. This strategy resulted in sub-atomic resolution MicroED data from proteinase K crystals, enabling accurate structure modelling and the visualization of detailed features. Interestingly, filtering out the noise revealed diffuse scattering phenomena that can hold additional structural information. Our findings suggest that combining energy filtering and electron counting can provide more accurate measurements at higher resolution, providing better insights into protein function and facilitating more precise model refinement.
History
DepositionSep 4, 2024-
Header (metadata) releaseMar 19, 2025-
Map releaseMar 19, 2025-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46871.jpg

Downloads & links

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Map

FileDownload / File: emd_46871.map.gz / Format: CCP4 / Size: 39.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicroED 2mFo-DFc map of proteinase K
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.27 Å/pix.
x 206 pix.
= 55.146 Å		0.27 Å/pix.
x 220 pix.
= 58.894 Å		0.27 Å/pix.
x 227 pix.
= 60.768 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: 0.2677 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.46
Minimum - Maximum-1.939028 - 19.006682999999999
Average (Standard dev.)-0.0026079651 (±0.97490627)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-50-120-179
Dimensions220227206
Spacing206220227
CellA: 55.1462 Å / B: 58.893997 Å / C: 60.7679 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

EntireName: Proteinase K
Components
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: CALCIUM ION
  • Ligand: NITRATE ION
  • 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: 28.958791 KDa
Recombinant expressionOrganism: Parengyodontium album (fungus)
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 LDIFGPGTDI 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: NITRATE ION

MacromoleculeName: NITRATE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: NO3
Molecular weightTheoretical: 62.005 Da
Chemical component information

ChemComp-NO3:
NITRATE ION

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 334 / 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

Concentration40 mg/mL
BufferpH: 6.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Details: Negative 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER
DetailsMicrocrystals

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Electron microscopy

MicroscopeTFS KRIOS
TemperatureMin: 77.0 K / Max: 90.0 K
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 420 / Average exposure time: 1.0 sec. / Average electron dose: 0.002 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Camera length: 1402 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 1.09 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: phenix.refine (ver. 1.21.1)
Molecular replacementSoftware - Name: PHASER (ver. 2.8.3)
Symmetry determination software listSoftware - Name: XDS (ver. BUILT=20230630)
Merging software listSoftware - Name: XSCALE (ver. BUILT=20230630)
Crystallography statisticsNumber intensities measured: 2811895 / Number structure factors: 98228 / Fourier space coverage: 97.5 / R merge: 28.4 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 1.09 Å / Shell - Shell ID: 1 / Shell - High resolution: 1.09 Å / Shell - Low resolution: 56.82 Å / Shell - Number structure factors: 98228 / Shell - Phase residual: 16.04 / Shell - Fourier space coverage: 97.5 / Shell - Multiplicity: 28.5

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Atomic model buiding 1

Initial modelPDB ID:

5kxv


Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: Molecular replacement
RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 11.29 / Target criteria: Maximum likelihood
Output model

PDB-9dho:
Structure of proteinase K from energy-filtered MicroED data

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