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- EMDB-48783: Structure of proteinase K from energy-filtered MicroED data using... -

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Basic information

Entry
Database: EMDB / ID: EMD-48783
TitleStructure of proteinase K from energy-filtered MicroED data using a 5 eV slit width
Map data
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 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. ...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: 1.2 Å
AuthorsClabbers MTB / Hattne J / Martynowycz 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: 2025
Title: Characterization of energy filtering slit widths for MicroED data collection.
Authors: Max T B Clabbers / Johan Hattne / Michael W Martynowycz / Tamir Gonen /
Abstract: A favorable signal-to-noise ratio is essential for obtaining high-quality diffraction data in macromolecular electron crystallography. Inelastic scattering contributes significantly to the noise, ...A favorable signal-to-noise ratio is essential for obtaining high-quality diffraction data in macromolecular electron crystallography. Inelastic scattering contributes significantly to the noise, reducing contrast between diffraction peaks and background, which complicates peak detection and compromises the accuracy of intensity integration. Energy filtering mitigates these challenges and enhances diffraction data quality by removing the inelastically scattered electrons, leading to reduced background noise and sharper Bragg peaks. Previously, we reported a substantial improvement in MicroED data quality and resolution with energy filtering. Here, we systematically evaluate the impact of different energy filter slit widths for optimal MicroED data collection. Data from proteinase K lamellae were collected using the 5, 10, and 20 eV energy filter slit widths. Our results show that the narrowest slit widths result in a stronger diffraction signal with lower background noise, improving the precision of the intensity measurements which resulted in better structural models. Our findings provide insights into the optimization of energy filter slit settings that, when paired with direct electron detection, enhance MicroED data collection strategies in MicroED by improving the signal-to-noise ratio, supporting higher quality data and ultimately enabling more precise structure determination.
History
DepositionJan 24, 2025-
Header (metadata) releaseJul 29, 2026-
Map releaseJul 29, 2026-
UpdateJul 29, 2026-
Current statusJul 29, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_48783.png

Downloads & links

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Map

FileDownload / File: emd_48783.map.gz / Format: CCP4 / Size: 31.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.28 Å/pix.
x 198 pix.
= 55.202 Å		0.28 Å/pix.
x 212 pix.
= 59.106 Å		0.28 Å/pix.
x 197 pix.
= 54.924 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: 0.2788 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.46
Minimum - Maximum-2.073125 - 17.245432000000001
Average (Standard dev.)-0.0071772984 (±0.97611725)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-48-102-172
Dimensions212197198
Spacing198212197
CellA: 55.2024 Å / B: 59.105602 Å / C: 54.923603 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
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: 5 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: 1.2 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: PHENIX (ver. 1.21.1)
CTF correctionType: NONE
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: 1000823 / Number structure factors: 75213 / Fourier space coverage: 97.9 / R merge: 25.1 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 1.2 Å / Shell - Shell ID: 1 / Shell - High resolution: 1.2 Å / Shell - Low resolution: 47.32 Å / Shell - Number structure factors: 75213 / Shell - Phase residual: 14.3 / Shell - Fourier space coverage: 97.9 / Shell - Multiplicity: 13.3

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Atomic model buiding 1

Initial modelPDB ID:

9dho


Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: Molecular replacement
RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 10.4 / Target criteria: Maximum likelihood
Output model

PDB-9n0f:
Structure of proteinase K from energy-filtered MicroED data using a 5 eV slit width

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