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- EMDB-4416: F97L counting mode -

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Basic information

Entry
Database: EMDB / ID: EMD-4416
TitleF97L counting mode
Map data
Sample
  • Virus: Hepatitis B virus
    • Protein or peptide: Hepatitis B virus core protein
Biological speciesHepatitis B virus
Methodsingle particle reconstruction / cryo EM / Resolution: 2.36 Å
AuthorsSong B / Flegler V / Makbul C / Rasmussen T / Bottcher B
CitationJournal: Ultramicroscopy / Year: 2019
Title: Capabilities of the Falcon III detector for single-particle structure determination.
Authors: Boyuan Song / Julian Lenhart / Vanessa Judith Flegler / Cihan Makbul / Tim Rasmussen / Bettina Böttcher /
Abstract: Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the ...Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the counting mode in which single electrons are counted, and the integrating mode in which the signal that arises from the incident electrons is integrated. While counting mode leads to far higher detective quantum efficiency at all spatial frequencies, the integrating mode enables faster data acquisition at higher exposure rates. For optimal throughput at best possible resolution it is important to understand when the better performance in counting mode becomes essential for solving a structure and when the lower detective quantum efficiency in integrating mode can be compensated by increasing the number of particles in the data set. Here, we provide a case study of the Falcon III camera, which has counting mode capability at exposure rates of <0.9 e/Px² and integrating mode capability at exposure rates above 10 e/Px². We found that counting mode gives better resolution for medium sized complexes such as the β-galactosidase (465 kDa) (2.2 Å, 97% of Nyquist vs. 2.4 Å, 89% of Nyquist) with data sets of similar size. However, for larger particles such as Hepatitis B virus capsid like particles (4.8 MDa) we did not find any resolution gain in counting mode.
History
DepositionNov 13, 2018-
Header (metadata) releaseFeb 20, 2019-
Map releaseFeb 20, 2019-
UpdateMay 1, 2019-
Current statusMay 1, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4416.png

Downloads & links

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Map

FileDownload / File: emd_4416.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.0635 Å
Density
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.21811146 - 0.36735728
Average (Standard dev.)0.00014155719 (±0.022200825)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 425.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06351.06351.0635
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z425.400425.400425.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.2180.3670.000

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Supplemental data

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Half map: #2

Fileemd_4416_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_4416_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hepatitis B virus

EntireName: Hepatitis B virus
Components
  • Virus: Hepatitis B virus
    • Protein or peptide: Hepatitis B virus core protein

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Supramolecule #1: Hepatitis B virus

SupramoleculeName: Hepatitis B virus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / Details: core protein was expressed in E.coli / NCBI-ID: 10407 / Sci species name: Hepatitis B virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: OTHER / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Homo sapiens (human)
Host systemOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 4.8 MDa
Virus shellShell ID: 1 / Diameter: 340.0 Å / T number (triangulation number): 4

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Macromolecule #1: Hepatitis B virus core protein

MacromoleculeName: Hepatitis B virus core protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Hepatitis B virus
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MDIDPYKEFG ATVELLSFLP SDFFPSVRDL LDTASALYRE ALESPEHCSP HHTALRQAIL CWGELMTLAT WVGVNLEDP ASRDLVVSYV NTNMGLKLRQ LLWFHISCLT FGRETVIEYL VSFGVWIRTP PAYRPPNAPI L STLPETTV VRRRGRSPRK RTPSPRKRRS QSPRRRRSQS RESQC

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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

Concentration4 mg/mL
BufferpH: 7.5 / Details: 25 mM Tris
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Quantifoil 300 mesh gold ultrafoil grids R 1.2/1.3 were used (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were glow discharged in air at a pressure of 2.2x10-2 Torr for 2 min at ...Details: Quantifoil 300 mesh gold ultrafoil grids R 1.2/1.3 were used (Quantifoil Micro Tools GmbH, Jena, Germany). Grids were glow discharged in air at a pressure of 2.2x10-2 Torr for 2 min at medium power with a Harrick Plasma cleaner (PDC-002). Subsequently, 3-4 ul of the sample was pipetted onto the glow discharged grids and plunge frozen in liquid Ethane with a Vitrobot IV (FEI). The sample chamber of the Vitrobot was maintained at 4C and 100% humidity. Wait time between sample application and blotting was 45 s, the drain time 0 s, the blot time 3 s and the blot force 0..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.57 µm / Calibrated defocus min: 0.68 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 79.0 K / Max: 80.0 K
Detailsdata has been recorded in counting mode 40 frames per movie
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1616 / Average exposure time: 75.0 sec. / Average electron dose: 40.0 e/Å2
Details: images were collected in movie mode (31 frames in total)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 88711
CTF correctionSoftware - Name: RELION
Startup modelType of model: EMDB MAP
EMDB ID:

0272


Details: previously determined
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: I (icosahedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.36 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 45444
DetailsMovies were motion corrected and exposure weighted with motioncor2. The defocus was determined for the motion corrected and exposure weighted averages with ctffind4
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 75

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