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- EMDB-23355: Structure of dNTPase at 3.3 Angstrom Resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-23355
TitleStructure of dNTPase at 3.3 Angstrom Resolution
Map data
SampledNTPase
Biological speciesVibrio cholerae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsBouvette J / Liu HF / Du X / Zhou Y / Sikkema AP / Mello JFR / Klemm B / Huang R / Schaaper RM / Borgnia MJ / Bartesaghi A
CitationJournal: Nat Commun / Year: 2021
Title: Beam image-shift accelerated data acquisition for near-atomic resolution single-particle cryo-electron tomography.
Authors: Jonathan Bouvette / Hsuan-Fu Liu / Xiaochen Du / Ye Zhou / Andrew P Sikkema / Juliana da Fonseca Rezende E Mello / Bradley P Klemm / Rick Huang / Roel M Schaaper / Mario J Borgnia / Alberto Bartesaghi /
Abstract: Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of ...Tomographic reconstruction of cryopreserved specimens imaged in an electron microscope followed by extraction and averaging of sub-volumes has been successfully used to derive atomic models of macromolecules in their biological environment. Eliminating biochemical isolation steps required by other techniques, this method opens up the cell to in-situ structural studies. However, the need to compensate for errors in targeting introduced during mechanical navigation of the specimen significantly slows down tomographic data collection thus limiting its practical value. Here, we introduce protocols for tilt-series acquisition and processing that accelerate data collection speed by up to an order of magnitude and improve map resolution compared to existing approaches. We achieve this by using beam-image shift to multiply the number of areas imaged at each stage position, by integrating geometrical constraints during imaging to achieve high precision targeting, and by performing per-tilt astigmatic CTF estimation and data-driven exposure weighting to improve final map resolution. We validated our beam image-shift electron cryo-tomography (BISECT) approach by determining the structure of a low molecular weight target (~300 kDa) at 3.6 Å resolution where density for individual side chains is clearly resolved.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionJan 26, 2021-
Header (metadata) releaseMar 3, 2021-
Map releaseMar 3, 2021-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_23355.map.gz / Format: CCP4 / Size: 3.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.37 Å/pix.
x 85 pix.
= 116.45 Å
1.37 Å/pix.
x 110 pix.
= 150.7 Å
1.37 Å/pix.
x 108 pix.
= 147.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.37 Å
Density
Contour LevelBy AUTHOR: 0.2 / Movie #1: 0.2
Minimum - Maximum-1.4229677 - 2.3423345
Average (Standard dev.)8.798084e-12 (±0.18561476)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin747786
Dimensions11010885
Spacing10811085
CellA: 147.96 Å / B: 150.7 Å / C: 116.45 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.371.371.37
M x/y/z10811085
origin x/y/z0.0000.0000.000
length x/y/z147.960150.700116.450
α/β/γ90.00090.00090.000
start NX/NY/NZ10510593
NX/NY/NZ143147156
MAP C/R/S123
start NC/NR/NS777486
NC/NR/NS10811085
D min/max/mean-1.4232.3420.000

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Supplemental data

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Segmentation: #1

Fileemd_23355_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Structure of dNTPase at 3.3 Angstrom Resolution

Fileemd_23355_additional_1.map
AnnotationStructure of dNTPase at 3.3 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Structure of dNTPase at 3.3 Angstrom Resolution

Fileemd_23355_half_map_1.map
AnnotationStructure of dNTPase at 3.3 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Structure of dNTPase at 3.3 Angstrom Resolution

Fileemd_23355_half_map_2.map
AnnotationStructure of dNTPase at 3.3 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire dNTPase

EntireName: dNTPase / Number of components: 1

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Component #1: protein, dNTPase

ProteinName: dNTPase / Recombinant expression: No
SourceSpecies: Vibrio cholerae (bacteria)
Source (engineered)Expression System: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.3 mg/mL / pH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 60 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 105000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: D3 (2x3 fold dihedral) / Number of projections: 34190
3D reconstructionAlgorithm: FOURIER SPACE / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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