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- EMDB-0343: MicroED structure of Proteinase K at 2.75A resolution from a sing... -

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Entry
Database: EMDB / ID: 0343
TitleMicroED structure of Proteinase K at 2.75A resolution from a single milled crystal.
Map dataMicroED 2Fo-Fc map of proteinase K at 2.75A resolution from a single milled crystal.
SampleProteinase K:
(ligand) x 3
Function / homologyProteinase K-like catalytic domain / Serine proteases, subtilase family, aspartic acid active site. / Serine proteases, subtilase family, serine active site. / Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase S8, subtilisin-related / Peptidase S8, subtilisin, His-active site / Peptidase S8, subtilisin, Asp-active site / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase family, histidine active site. ...Proteinase K-like catalytic domain / Serine proteases, subtilase family, aspartic acid active site. / Serine proteases, subtilase family, serine active site. / Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase S8, subtilisin-related / Peptidase S8, subtilisin, His-active site / Peptidase S8, subtilisin, Asp-active site / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase family, histidine active site. / Peptidase S8/S53 domain superfamily / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Subtilase family / Peptidase inhibitor I9 / peptidase K / serine-type endopeptidase activity / metal ion binding / Proteinase K
Function and homology information
SourceEngyodontium album (fungus)
Methodelectron crystallography / cryo EM / 2.75 Å resolution
AuthorsMartynowycz MW / Zhao W / Hattne J / Jensen GJ / Gonen T
CitationJournal: Structure / Year: 2018
Title: Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size.
Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen
Abstract: Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation ...Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.
Validation ReportPDB-ID: 6n4u

SummaryFull reportAbout validation report
DateDeposition: Nov 20, 2018 / Header (metadata) release: Feb 6, 2019 / Map release: Feb 6, 2019 / Last update: Feb 6, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.46937
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 1.46937
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6n4u
  • Surface level: 1.46937
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0343.map.gz (map file in CCP4 format, 1707 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
72 pix
0.67 Å/pix.
= 67.46 Å
79 pix
0.67 Å/pix.
= 67.46 Å
75 pix
0.67 Å/pix.
= 106.67 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

(generated in cubic-lattice coordinate)

Voxel sizeX: 0.6746 Å / Y: 0.6746 Å / Z: 0.66669 Å
Density
Contour Level:1.46937 (by author), 1.46937 (movie #1):
Minimum - Maximum-3.9357493 - 5.0805335
Average (Standard dev.)0.00043542887 (0.97957665)
Details

EMDB XML:

Space Group Number96
Map Geometry
Axis orderZYX
Dimensions797572
Origin-63.0-38.0-16.0
Limit15.036.055.0
Spacing100100160
CellA: 67.46 Å / B: 67.46 Å / C: 106.67 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.67460.67460.6666875
M x/y/z100100160
origin x/y/z0.0000.0000.000
length x/y/z67.46067.460106.670
α/β/γ90.00090.00090.000
start NX/NY/NZ-16-63-38
NX/NY/NZ727975
MAP C/R/S321
start NC/NR/NS-38-63-16
NC/NR/NS757972
D min/max/mean-3.9365.0810.000

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Supplemental data

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Sample components

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Entire Proteinase K

EntireName: Proteinase K / Number of components: 5

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Component #1: cellular-component, Proteinase K

Cellular-componentName: Proteinase K / Recombinant expression: No
MassTheoretical: 28.93 kDa
SourceSpecies: Engyodontium album (fungus)

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Component #2: protein, Proteinase K

ProteinName: Proteinase K / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 28.958791 kDa
SourceSpecies: Engyodontium album (fungus)
Source (engineered)Expression System: Engyodontium album (fungus)

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Component #3: ligand, CALCIUM ION

LigandName: CALCIUM IONCalcium / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 4.007805 MDa

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Component #4: ligand, SULFATE ION

LigandName: SULFATE IONSulfate / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.606305 MDa

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Component #5: ligand, water

LigandName: water / Number of Copies: 23 / Recombinant expression: No
MassTheoretical: 1.801505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: 3D array / Method: cryo EM
Crystal parametersSpace group: P43 21 2 / A: 67.46 Å / B: 67.46 Å / C: 106.67 Å / Alpha: 90 deg. / Beta: 90 deg. / Gamma: 90 deg.
Sample solutionSpecimen conc.: 20 mg/ml / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 273 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 0.04 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: DIFFRACTION
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 77.0 - 90.0 K)
CameraDetector: FEI CETA (4k x 4k)

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Image acquisition

Image acquisitionSampling size: 28 microns
Details: Continuous rotation from -30 to +30 with a rotation rate of 0.2 degrees per second and a readout every 3 seconds.

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Image processing

ProcessingMethod: electron crystallography / Details: This was the new CetaD.
3D reconstructionCTF correction: NONE / Resolution: 2.75 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: R / Refinement space: RECIPROCAL
Input PDB model: 5I9S
Overall bvalue: 21.100000000000001
Output model

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