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- EMDB-25468: MicroED structure of proteinase K from a 320 nm thick lamella mea... -

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Basic information

Entry
Database: EMDB / ID: EMD-25468
TitleMicroED structure of proteinase K from a 320 nm thick lamella measured at 300 kV
Map data2Fo-Fc MicroED map from a 320 nm thick lamella measured at 300 kV
Sample
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • 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. / 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
AuthorsMartynowycz MW / Clabbers MTB
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Benchmarking the ideal sample thickness in cryo-EM.
Authors: Michael W Martynowycz / Max T B Clabbers / Johan Unge / Johan Hattne / Tamir Gonen /
Abstract: The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K ...The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.
History
DepositionNov 19, 2021-
Header (metadata) releaseSep 7, 2022-
Map releaseSep 7, 2022-
UpdateOct 18, 2023-
Current statusOct 18, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25468.png

Downloads & links

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Map

FileDownload / File: emd_25468.map.gz / Format: CCP4 / Size: 4.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2Fo-Fc MicroED map from a 320 nm thick lamella measured at 300 kV
Voxel sizeX: 0.50622 Å / Y: 0.50622 Å / Z: 0.48667 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-3.6755447 - 5.5821853
Average (Standard dev.)-0.0026863958 (±0.9810498)
SymmetrySpace group: 96
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-89-56-6
Dimensions11110694
Spacing135135216
CellA: 68.34 Å / B: 68.34 Å / C: 105.12 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: 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: 28.930783 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: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 101 / 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

Concentration5 mg/mL
BufferpH: 7.5
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: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER
DetailsMicrocrystals

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Camera length: 2460 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 77.0 K / Max: 90.0 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: 1 / Number diffraction images: 120 / Average exposure time: 1.0 sec. / Average electron dose: 0.01 e/Å2
Details: 0.5 degrees per second, 1 second readout, 30 to -30 degrees.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Crystallography statisticsNumber intensities measured: 71235 / Number structure factors: 22907 / Fourier space coverage: 83 / R sym: 14 / R merge: 30 / Overall phase error: 25 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 2.1 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.1 Å / Shell - Low resolution: 2.18 Å / Shell - Number structure factors: 2161 / Shell - Phase residual: 34 / Shell - Fourier space coverage: 84 / Shell - Multiplicity: 3
Final reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: PHENIX
Merging software listSoftware - Name: AIMLESS
DetailsBinned by 2.

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Atomic model buiding 1

Initial modelPDB ID:

6cl7


Chain - Chain ID: A / Chain - Residue range: 106-384 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 19 / Target criteria: Maximum likelihood
Output model

PDB-7swa:
MicroED structure of proteinase K from a 320 nm thick lamella measured at 300 kV

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