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- PDB-9n0f: Structure of proteinase K from energy-filtered MicroED data using... -

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Basic information

Entry
Database: PDB / ID: 9n0f
TitleStructure of proteinase K from energy-filtered MicroED data using a 5 eV slit width
ComponentsProteinase K
KeywordsHYDROLASE / serine protease
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
NITRATE ION / Proteinase K
Similarity search - Component
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.2 Å
AuthorsClabbers, M.T.B. / Hattne, J. / Martynowycz, M.W. / Gonen, T.
Funding support United States, 3items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 29, 2026Provider: repository / Type: Initial release
Revision 1.0Jul 29, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jul 29, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jul 29, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,1014
Polymers28,9591
Non-polymers1423
Water6,017334
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)66.920, 66.920, 107.560
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Protein Proteinase K / Endopeptidase K / Tritirachium alkaline proteinase


Mass: 28958.791 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Parengyodontium album (fungus) / Gene: PROK / Production host: Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-NO3 / NITRATE ION


Mass: 62.005 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: NO3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 334 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Proteinase K / Type: COMPLEX / Details: Serine protease / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.0289 MDa / Experimental value: NO
Source (natural)Organism: Parengyodontium album (fungus)
Source (recombinant)Organism: Parengyodontium album (fungus)
Buffer solutionpH: 6.5
SpecimenConc.: 40 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Microcrystals
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Data collection

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingAverage exposure time: 1 sec. / Electron dose: 0.002 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 420 / Num. of grids imaged: 1 / Num. of real images: 1
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 5 eV
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096
EM diffractionCamera length: 1402 mm
EM diffraction shellResolution: 1.2→47.32 Å / Fourier space coverage: 97.9 % / Multiplicity: 13.3 / Num. of structure factors: 75213 / Phase residual: 14.3 °
EM diffraction statsFourier space coverage: 97.9 % / High resolution: 1.2 Å / Num. of intensities measured: 1000823 / Num. of structure factors: 75213 / Phase error rejection criteria: None / Rmerge: 25.1
ReflectionBiso Wilson estimate: 8.82 Å2

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Processing

EM software
IDNameVersionCategory
1SerialEM4.2.0image acquisition
6Coot0.9.8.93model fitting
8PHASER2.8.3molecular replacement
10XDSBUILT=20230630symmetry determination
11XSCALEBUILT=20230630crystallography merging
12PHENIX1.21.13D reconstruction
13PHENIX1.21.1model refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 66.92 Å / B: 66.92 Å / C: 107.56 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 1.2 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 10.4 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: Maximum likelihood
Atomic model buildingPDB-ID: 9dho

9dho


Accession code: 9dho / Details: Molecular replacement / Source name: PDB / Type: experimental model
RefinementResolution: 1.2→47.32 Å / SU ML: 0.0889 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 14.3408
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.1696 3769 5.01 %
Rwork0.1533 71444 -
obs0.1541 75213 97.81 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 10.4 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.01542267
ELECTRON CRYSTALLOGRAPHYf_angle_d1.21033113
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.1135337
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0125426
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d12.1798801
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.2-1.220.2571290.25652609ELECTRON CRYSTALLOGRAPHY98
1.22-1.230.24981210.24372635ELECTRON CRYSTALLOGRAPHY98.01
1.23-1.250.25091310.24132595ELECTRON CRYSTALLOGRAPHY97.81
1.25-1.270.21881370.22422627ELECTRON CRYSTALLOGRAPHY97.77
1.27-1.280.23511510.22832567ELECTRON CRYSTALLOGRAPHY97.95
1.28-1.30.26981410.22292623ELECTRON CRYSTALLOGRAPHY97.98
1.3-1.330.23381440.22042620ELECTRON CRYSTALLOGRAPHY97.91
1.33-1.350.23981270.21472625ELECTRON CRYSTALLOGRAPHY97.97
1.35-1.370.26941290.20482634ELECTRON CRYSTALLOGRAPHY98.22
1.37-1.40.2281510.20782603ELECTRON CRYSTALLOGRAPHY97.97
1.4-1.430.18011460.18692617ELECTRON CRYSTALLOGRAPHY98.12
1.43-1.460.20651250.18142639ELECTRON CRYSTALLOGRAPHY97.91
1.46-1.490.19141300.17372637ELECTRON CRYSTALLOGRAPHY97.84
1.49-1.530.17661080.1722658ELECTRON CRYSTALLOGRAPHY98.02
1.53-1.570.18491400.16822626ELECTRON CRYSTALLOGRAPHY98.12
1.57-1.620.17171750.16312593ELECTRON CRYSTALLOGRAPHY97.98
1.62-1.670.191430.15782654ELECTRON CRYSTALLOGRAPHY98
1.67-1.730.16771340.15192627ELECTRON CRYSTALLOGRAPHY97.73
1.73-1.80.17081390.14672647ELECTRON CRYSTALLOGRAPHY97.79
1.8-1.880.14271370.13132660ELECTRON CRYSTALLOGRAPHY98
1.88-1.980.13721460.12452659ELECTRON CRYSTALLOGRAPHY97.97
1.98-2.110.12731390.11572669ELECTRON CRYSTALLOGRAPHY97.64
2.11-2.270.12751380.11322668ELECTRON CRYSTALLOGRAPHY97.74
2.27-2.50.1371630.12252668ELECTRON CRYSTALLOGRAPHY97.72
2.5-2.860.15061330.12952698ELECTRON CRYSTALLOGRAPHY97.49
2.86-3.60.15411430.12512737ELECTRON CRYSTALLOGRAPHY97.17
3.6-47.320.15541690.1442849ELECTRON CRYSTALLOGRAPHY96.33

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