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- EMDB-25185: Ab initio structure of proteinase K from electron-counted MicroED data -

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Basic information

Entry
Database: EMDB / ID: EMD-25185
TitleAb initio structure of proteinase K from electron-counted MicroED data
Map data1.5A resolution 2mFo-dFc MicroED map of proteinase K determined ab initio by electron-counted data
Sample
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid
  • Ligand: CALCIUM IONCalcium
  • Ligand: water
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. / 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) / Engyodontium album, Tritirachium album
Methodelectron crystallography / cryo EM
AuthorsMartynowycz MW / Clabbers MTB / Hattne J / Gonen T
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: Nat Methods / Year: 2022
Title: Ab initio phasing macromolecular structures using electron-counted MicroED data.
Authors: Michael W Martynowycz / Max T B Clabbers / Johan Hattne / Tamir Gonen /
Abstract: Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals ...Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data.
History
DepositionOct 21, 2021-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25185.png

Downloads & links

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Map

FileDownload / File: emd_25185.map.gz / Format: CCP4 / Size: 10.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation1.5A resolution 2mFo-dFc MicroED map of proteinase K determined ab initio by electron-counted data
Voxel sizeX: 0.37267 Å / Y: 0.37267 Å / Z: 0.37076 Å
Density
Contour LevelBy AUTHOR: 1.5
Minimum - Maximum-2.7613127 - 12.3482485
Average (Standard dev.)-0.0034129387 (±0.9709549)
SymmetrySpace group: 96
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-30-6953
Dimensions147134137
Spacing180180288
CellA: 67.08 Å / B: 67.08 Å / C: 106.78 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: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid
  • 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 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: Engyodontium album, Tritirachium album
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

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Macromolecule #2: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid

MacromoleculeName: 5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid / type: ligand / ID: 2 / Number of copies: 2 / Formula: I3C
Molecular weightTheoretical: 558.835 Da
Chemical component information

ChemComp-I3C:
5-amino-2,4,6-triiodobenzene-1,3-dicarboxylic acid

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Macromolecule #3: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 234 / 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: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: NEGATIVE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER
DetailsMilled microcrystals

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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: 1738 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: -30.0, 30.0
TemperatureMin: 77.0 K / Max: 90.0 K
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Sampling interval: 28.0 µm / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 840 / Average exposure time: 0.5 sec. / Average electron dose: 0.001 e/Å2
Details: 0.15 degrees per second, 0.5 second readout, 30 to -30 degrees
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit cell - A: 67.08 Å / Unit cell - B: 67.08 Å / Unit cell - C: 106.78 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Space group: P 43 21 2
Crystallography statisticsNumber intensities measured: 416133 / Number structure factors: 39303 / Fourier space coverage: 98.87 / R sym: 0.087 / R merge: 0.277 / Overall phase error: 20 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 1.5 Å
Details: Phases were determined by placing 4 ideal helix fragments. These were extended by chain tracing and density modifications.
Shell - Shell ID: 1 / Shell - High resolution: 1.53 Å / Shell - Low resolution: 1.5 Å / Shell - Number structure factors: 1758 / Shell - Phase residual: 30 / Shell - Fourier space coverage: 89.3 / Shell - Multiplicity: 8.9
Final reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: REFMAC
Merging software listSoftware - Name: AIMLESS
DetailsBinned by 2.

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 14.137 / Target criteria: Maximum likelihood
Output model

PDB-7skx:
Ab initio structure of proteinase K from electron-counted MicroED data

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