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- EMDB-14407: Single particle structure of keyhole limpet hemocyanin obtained v... -

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Basic information

Entry
Database: EMDB / ID: EMD-14407
TitleSingle particle structure of keyhole limpet hemocyanin obtained via iDPC scanning transmission electron microscopy
Map datasharpened map from cryosparc local refinement v3.3.1
Sample
  • Complex: keyhole limpet hemocyanin
    • Protein or peptide: keyhole limpet hemocyanin
Biological speciesMegathura crenulata (invertebrata)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.51 Å
AuthorsMann D / Lazic I / Wirix M / de Haas F / Sachse C
Funding support Germany, 1 items
OrganizationGrant numberCountry
Helmholtz Association Germany
CitationJournal: Nat Methods / Year: 2022
Title: Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution.
Authors: Ivan Lazić / Maarten Wirix / Max Leo Leidl / Felix de Haas / Daniel Mann / Maximilian Beckers / Evgeniya V Pechnikova / Knut Müller-Caspary / Ricardo Egoavil / Eric G T Bosch / Carsten Sachse /
Abstract: In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently ...In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution three-dimensional structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) and tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.
History
DepositionFeb 22, 2022-
Header (metadata) releaseJan 11, 2023-
Map releaseJan 11, 2023-
UpdateJan 11, 2023-
Current statusJan 11, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14407.png

Downloads & links

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Map

FileDownload / File: emd_14407.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map from cryosparc local refinement v3.3.1
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
2.44 Å/pix.
x 256 pix.
= 624.64 Å		2.44 Å/pix.
x 256 pix.
= 624.64 Å		2.44 Å/pix.
x 256 pix.
= 624.64 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.44 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-0.62414944 - 2.1362143
Average (Standard dev.)-0.0010584741 (±0.17536116)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 624.64 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14407_msk_1.map
Projections & Slices
AxesZYX

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Histogram

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Half map: half map A

Fileemd_14407_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_14407_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : keyhole limpet hemocyanin

EntireName: keyhole limpet hemocyanin
Components
  • Complex: keyhole limpet hemocyanin
    • Protein or peptide: keyhole limpet hemocyanin

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Supramolecule #1: keyhole limpet hemocyanin

SupramoleculeName: keyhole limpet hemocyanin / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Megathura crenulata (invertebrata)

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Macromolecule #1: keyhole limpet hemocyanin

MacromoleculeName: keyhole limpet hemocyanin / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Megathura crenulata (invertebrata)
SequenceString: ...String:

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: OTHER / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: OTHER / Average electron dose: 35.0 e/Å2
Details: STEM imaging was conducted using a Thermo Fisher Scientific Titan Krios G4, operated at 300 kV. The column was equipped with a standard high-brightness field emission gun (XFEG), three- ...Details: STEM imaging was conducted using a Thermo Fisher Scientific Titan Krios G4, operated at 300 kV. The column was equipped with a standard high-brightness field emission gun (XFEG), three-condenser lens system, C-TWIN objective lens with wide-gap pole piece (11 mm and Cs = 2.7mm), Panther segmented STEM detector, a Ceta camera and Falcon 4 direct electron detection (DED) camera. A combination of different C2 apertures (20 mum and 50 mum) and C2/C3 lens current ratios were used to create different CSA of the beam. Alignment for STEM was done by aligning beam shift, beam tilt pivot points and beam tilt in STEM mode for the different convergence angles. For accurate determination of the COM, a de-scan alignment has additionally been performed. The CSA of the applied beams were measured with high precision using the Au cross-grating and Ceta camera by first recording the radii of the gold diffraction rings for calibration and subsequently measuring the radius of the bright-field (BF) disc.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: D5 (2x5 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.51 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 18597
DetailsPanther segmented STEM detector
FSC plot (resolution estimation)

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