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- EMDB-3782: A 3.4 Angstrom structure of HIV-1 CA-SP1 by 3D-CTF correction of ... -

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Basic information

Entry
Database: EMDB / ID: EMD-3782
TitleA 3.4 Angstrom structure of HIV-1 CA-SP1 by 3D-CTF correction of cryo-electron tomograms
Map dataNone
Sample
  • Virus: Human immunodeficiency virus 1
Biological speciesHuman immunodeficiency virus 1
Methodsubtomogram averaging / cryo EM / Resolution: 3.4 Å
AuthorsTuronova B / Schur FKM / Wan W / Briggs JAG
CitationJournal: J Struct Biol / Year: 2017
Title: Efficient 3D-CTF correction for cryo-electron tomography using NovaCTF improves subtomogram averaging resolution to 3.4Å.
Authors: Beata Turoňová / Florian K M Schur / William Wan / John A G Briggs /
Abstract: Cryo-electron tomography (cryo-ET) allows cellular ultrastructures and macromolecular complexes to be imaged in three-dimensions in their native environments. Cryo-electron tomograms are ...Cryo-electron tomography (cryo-ET) allows cellular ultrastructures and macromolecular complexes to be imaged in three-dimensions in their native environments. Cryo-electron tomograms are reconstructed from projection images taken at defined tilt-angles. In order to recover high-resolution information from cryo-electron tomograms, it is necessary to measure and correct for the contrast transfer function (CTF) of the microscope. Most commonly, this is performed using protocols that approximate the sample as a two-dimensional (2D) plane. This approximation accounts for differences in defocus and therefore CTF across the tilted sample. It does not account for differences in defocus of objects at different heights within the sample; instead, a 3D approach is required. Currently available approaches for 3D-CTF correction are computationally expensive and have not been widely implemented. Here we simulate the benefits of 3D-CTF correction for high-resolution subtomogram averaging, and present a user-friendly, computationally-efficient 3D-CTF correction tool, NovaCTF, that is compatible with standard tomogram reconstruction workflows in IMOD. We validate the approach on synthetic data and test it using subtomogram averaging of real data. Consistent with our simulations, we find that 3D-CTF correction allows high-resolution structures to be obtained with much smaller subtomogram averaging datasets than are required using 2D-CTF. We also show that using equivalent dataset sizes, 3D-CTF correction can be used to obtain higher-resolution structures. We present a 3.4Å resolution structure determined by subtomogram averaging.
History
Header (metadata) releaseJun 29, 2016-
DepositionJul 5, 2017-
Map releaseAug 2, 2017-
UpdateSep 27, 2017-
Current statusSep 27, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.275
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.275
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.275
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3782.png

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Map

FileDownload / File: emd_3782.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.275 / Movie #1: 0.275
Minimum - Maximum-0.60958225 - 0.9421653
Average (Standard dev.)0.020314952 (±0.10203436)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z259.200259.200259.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.6100.9420.020

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Supplemental data

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Sample components

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Entire : Human immunodeficiency virus 1

EntireName: Human immunodeficiency virus 1
Components
  • Virus: Human immunodeficiency virus 1

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Supramolecule #1: Human immunodeficiency virus 1

SupramoleculeName: Human immunodeficiency virus 1 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Virus-like particles were obtained by in vitro assembly of a truncated Gag construct (deltaMACANCSP2) in presence of the maturation inhibitor Bevirimat
NCBI-ID: 11676 / Sci species name: Human immunodeficiency virus 1 / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: Yes
Host (natural)Organism: Homo sapiens (human)
Host systemOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET11C

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
50.0 mMHEPES
100.0 mMSodium ChlorideNaClSodium chloride
1.0 mMEDTAEthylenediaminetetraacetic acid
1.0 mMTCEP

Details: Virus-like particles were assembled in the presence of nucleic acid (73mer oligonucleotide, 1:10 molar ratio oligonucleotide:protein).
GridModel: C-flat / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Details: at 20 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK II
Details: 10nM colloidal gold was added to the sample prior to plunge freezing..
DetailsVirus-like particles were assembled in vitro

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsNanoprobe
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 8-10 / Number grids imaged: 2 / Average exposure time: 1.0 sec. / Average electron dose: 3.4 e/Å2
Details: Number of frames ranged from 8-10 Exposure time per tilt ranged from 0.8 to 1.0 seconds
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

ExtractionNumber tomograms: 43 / Number images used: 527528
Details: Subtomograms were extracted from the surface of each particle according to the determined radius of the particle.
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4)CTF determination
NOVACTFCTF-correction by multiplication and astigmatism correction

Details: 3D CTF-correction was performed using NOVACTF. Defocus step 15nm.
Final angle assignmentType: OTHER / Software: (Name: AV3, TOM Toolbox) / Details: Cross-correlation based template matching
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software: (Name: AV3, TOM Toolbox) / Number subtomograms used: 128773
DetailsFrames were aligned using MotionCorr. Tilts in a tilt series were exposure filtered for cumulative electron dose. Tomograms were reconstructed using IMOD.

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