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- EMDB-20365: MicroED structure of proteinase K from low-dose merged lamellae t... -

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Basic information

Entry
Database: EMDB / ID: EMD-20365
TitleMicroED structure of proteinase K from low-dose merged lamellae that were not pre-coated with platinum 2.16A resolution (LD)
Map dataMicroED 2Fo-Fc map from a low-dose merge of uncoated, polished lamellae of proteinase K crystals at a resolution of 2.16A (LD)
Sample
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: water
KeywordsHydrolase
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: 2.16 Å
AuthorsMartynowycz MW / Zhao W
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R35 GM122588 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)2P50GM082545 United States
CitationJournal: Structure / Year: 2019
Title: Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED.
Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction ...Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.
History
DepositionJun 29, 2019-
Header (metadata) releaseJul 24, 2019-
Map releaseSep 4, 2019-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4155
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.4155
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6pks
  • Surface level: 0.4155
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20365.png

Downloads & links

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Map

FileDownload / File: emd_20365.map.gz / Format: CCP4 / Size: 33.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicroED 2Fo-Fc map from a low-dose merge of uncoated, polished lamellae of proteinase K crystals at a resolution of 2.16A (LD)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.33 Å/pix.
x 215 pix.
= 70.795 Å		0.35 Å/pix.
x 201 pix.
= 70.633 Å		0.35 Å/pix.
x 201 pix.
= 70.633 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.35141 Å / Y: 0.35141 Å / Z: 0.32928 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4155 / Movie #1: 0.4155
Minimum - Maximum-0.6878314 - 1.4456935
Average (Standard dev.)0.00021253635 (±0.27699566)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-40-149-107
Dimensions201201215
Spacing201201215
CellA: 70.63265 Å / B: 70.63265 Å / C: 70.79546 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.3514079601990.3514079601990.32927906976744
M x/y/z201201215
origin x/y/z0.0000.0000.000
length x/y/z70.63370.63370.795
α/β/γ90.00090.00090.000
start NX/NY/NZ-40-149-107
NX/NY/NZ201201215
MAP C/R/S213
start NC/NR/NS-149-40-107
NC/NR/NS201201215
D min/max/mean-0.6881.4460.000

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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
Source (natural)Organism: Parengyodontium album (fungus)
Molecular weightTheoretical: 28.93 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: 55 / 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

Concentration20 mg/mL
BufferpH: 7.5
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 273.0 K / Instrument: FEI VITROBOT MARK IV
DetailsProteinase K purchased from Sigma.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 77.0 K / Max: 90.0 K
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 100 / Number diffraction images: 100 / Average exposure time: 3.0 sec. / Average electron dose: 0.03 e/Å2
Details: Continuous rotation with a rotation rate of 0.2 degrees per second and a readout every 3 seconds
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 2055.0 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

DetailsThis was the new CetaD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.16 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: PHENIX (ver. 1.15.2)
Molecular replacementSoftware - Name: PHENIX (ver. 2.8.2)
Crystallography statisticsNumber intensities measured: 126914 / Number structure factors: 13527 / Fourier space coverage: 98.87 / R sym: 0.47 / R merge: 0.45 / Overall phase error: 26.59 / Overall phase residual: 26.59 / Phase error rejection criteria: 0 / High resolution: 2.16 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.16 Å / Shell - Low resolution: 41.52 Å / Shell - Number structure factors: 13527 / Shell - Phase residual: 26.59 / Shell - Fourier space coverage: 98.87 / Shell - Multiplicity: 9.4

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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: OTHER / Overall B value: 34.2
Output model

PDB-6pks:
MicroED structure of proteinase K from low-dose merged lamellae that were not pre-coated with platinum 2.16A resolution (LD)

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