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

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Basic information

Entry
Database: EMDB / ID: EMD-0343
TitleMicroED structure of Proteinase K at 2.75A resolution from a single milled crystal.
Map data
SampleProteinase K:
(ligand) x 3
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / metal ion binding
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 / Proteinase K-like catalytic domain / Peptidase S8/S53 domain superfamily / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily
Proteinase K
Biological speciesEngyodontium album (fungus)
Methodelectron crystallography / cryo EM / Resolution: 2.75 Å
AuthorsMartynowycz MW / Zhao W / Hattne J / Jensen GJ / Gonen T
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R35 GM122588 United States
CitationJournal: Structure / Year: 2019
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
History
DepositionNov 20, 2018-
Header (metadata) releaseFeb 6, 2019-
Map releaseFeb 6, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: RCSB / Status: Released

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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

FileDownload / File: emd_0343.map.gz / Format: CCP4 / Size: 1.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.67 Å/pix.
x 72 pix.
= 67.46 Å
0.67 Å/pix.
x 79 pix.
= 67.46 Å
0.67 Å/pix.
x 75 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 LevelBy AUTHOR: 1.46937 / Movie #1: 1.46937
Minimum - Maximum-3.9357493 - 5.0805335
Average (Standard dev.)0.00043542887 (±0.97957665)
SymmetrySpace group: 96
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-63-38-16
Dimensions797572
Spacing100100160
CellA: 67.46 Å / B: 67.46 Å / C: 106.67 Å
α=β=γ: 90.0 °

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: P43212 / A: 67.46 Å / B: 67.46 Å / C: 106.67 Å / α: 90 %deg; / β: 90 %deg; / γ: 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: (77.0 - 90.0 K)
CameraDetector: FEI CETA (4k x 4k)

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Image acquisition

Image acquisitionSampling size: 28 µm
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: 21.1
Output model

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