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- EMDB-23356: Structure of dNTPase at 3.6 Angstrom Resolution -

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Basic information

Entry
Database: EMDB / ID: EMD-23356
TitleStructure of dNTPase at 3.6 Angstrom Resolution
Map dataStructure of dNTPase at 3.6 Angstrom Resolution
Sample
  • Complex: dNTPase
Function / homology
Function and homology information


dGTPase activity / dGTP catabolic process
Similarity search - Function
dNTP triphosphohydrolase, type 2 / Phosphohydrolase-associated domain / Phosphohydrolase-associated domain / dNTP triphosphohydrolase / HD domain profile. / HD domain / Metal dependent phosphohydrolases with conserved 'HD' motif. / HD/PDEase domain
Similarity search - Domain/homology
Deoxyguanosinetriphosphate triphosphohydrolase-like protein 1
Similarity search - Component
Biological speciesVibrio cholerae (bacteria)
Methodsubtomogram averaging / cryo EM / Resolution: 3.6 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.
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: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23356.png

Downloads & links

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Map

FileDownload / File: emd_23356.map.gz / Format: CCP4 / Size: 2.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of dNTPase at 3.6 Angstrom Resolution
Voxel sizeX=Y=Z: 1.37 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-1.1884931 - 2.0925255
Average (Standard dev.)-3.4805915e-12 (±0.20059966)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin515663
Dimensions999673
Spacing969973
CellA: 131.52 Å / B: 135.63 Å / C: 100.01 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.371.371.37
M x/y/z969973
origin x/y/z0.0000.0000.000
length x/y/z131.520135.630100.010
α/β/γ90.00090.00090.000
start NX/NY/NZ10510593
NX/NY/NZ143147156
MAP C/R/S123
start NC/NR/NS565163
NC/NR/NS969973
D min/max/mean-1.1882.093-0.000

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Supplemental data

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Mask #1

Fileemd_23356_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Structure of dNTPase at 3.6 Angstrom Resolution

Fileemd_23356_additional_1.map
AnnotationStructure of dNTPase at 3.6 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Structure of dNTPase at 3.6 Angstrom Resolution

Fileemd_23356_half_map_1.map
AnnotationStructure of dNTPase at 3.6 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Structure of dNTPase at 3.6 Angstrom Resolution

Fileemd_23356_half_map_2.map
AnnotationStructure of dNTPase at 3.6 Angstrom Resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : dNTPase

EntireName: dNTPase
Components
  • Complex: dNTPase

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Supramolecule #1: dNTPase

SupramoleculeName: dNTPase / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Vibrio cholerae (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration1.3 mg/mL
BufferpH: 7.5
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Average electron dose: 5.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 64 / Number images used: 34435 / Method: Manually picked
Final 3D classificationNumber classes: 1
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 34435
FSC plot (resolution estimation)

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