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- EMDB-4414: beta-galactosidase (integrating mode) -

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Basic information

Entry
Database: EMDB / ID: EMD-4414
Titlebeta-galactosidase (integrating mode)
Map data
Sample
  • Complex: beta-galactosidase
    • Protein or peptide: beta-galactosidase
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsSong B / Flegler V
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation359471283 Germany
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) releaseNov 28, 2018-
Map releaseFeb 20, 2019-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4414.png

Downloads & links

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Map

FileDownload / File: emd_4414.map.gz / Format: CCP4 / Size: 824 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: 0.06
Minimum - Maximum-0.15604354 - 0.37473986
Average (Standard dev.)0.0000461827 (±0.008211129)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 638.10004 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.06351.06351.0635
M x/y/z600600600
origin x/y/z0.0000.0000.000
length x/y/z638.100638.100638.100
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ320320320
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS600600600
D min/max/mean-0.1560.3750.000

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Supplemental data

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Half map: #2

Fileemd_4414_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_4414_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 : beta-galactosidase

EntireName: beta-galactosidase
Components
  • Complex: beta-galactosidase
    • Protein or peptide: beta-galactosidase

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Supramolecule #1: beta-galactosidase

SupramoleculeName: beta-galactosidase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: sample was purchased from Sigma G5635
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 465 KDa

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Macromolecule #1: beta-galactosidase

MacromoleculeName: beta-galactosidase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: beta-galactosidase
SequenceString: MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEA VPESWLECDL PEADTVVVPS NWQMHGYDAP IYTNVTYPIT VNPPFVPTEN P TGCYSLTF NVDESWLQEG QTRIIFDGVN SAFHLWCNGR WVGYGQDSRL ...String:
MTMITDSLAV VLQRRDWENP GVTQLNRLAA HPPFASWRNS EEARTDRPSQ QLRSLNGEWR FAWFPAPEA VPESWLECDL PEADTVVVPS NWQMHGYDAP IYTNVTYPIT VNPPFVPTEN P TGCYSLTF NVDESWLQEG QTRIIFDGVN SAFHLWCNGR WVGYGQDSRL PSEFDLSAFL RA GENRLAV MVLRWSDGSY LEDQDMWRMS GIFRDVSLLH KPTTQISDFH VATRFNDDFS RAV LEAEVQ MCGELRDYLR VTVSLWQGET QVASGTAPFG GEIIDERGGY ADRVTLRLNV ENPK LWSAE IPNLYRAVVE LHTADGTLIE AEACDVGFRE VRIENGLLLL NGKPLLIRGV NRHEH HPLH GQVMDEQTMV QDILLMKQNN FNAVRCSHYP NHPLWYTLCD RYGLYVVDEA NIETHG MVP MNRLTDDPRW LPAMSERVTR MVQRDRNHPS VIIWSLGNES GHGANHDALY RWIKSVD PS RPVQYEGGGA DTTATDIICP MYARVDEDQP FPAVPKWSIK KWLSLPGETR PLILCEYA H AMGNSLGGFA KYWQAFRQYP RLQGGFVWDW VDQSLIKYDE NGNPWSAYGG DFGDTPNDR QFCMNGLVFA DRTPHPALTE AKHQQQFFQF RLSGQTIEVT SEYLFRHSDN ELLHWMVALD GKPLASGEV PLDVAPQGKQ LIELPELPQP ESAGQLWLTV RVVQPNATAW SEAGHISAWQ Q WRLAENLS VTLPAASHAI PHLTTSEMDF CIELGNKRWQ FNRQSGFLSQ MWIGDKKQLL TP LRDQFTR APLDNDIGVS EATRIDPNAW VERWKAAGHY QAEAALLQCT ADTLADAVLI TTA HAWQHQ GKTLFISRKT YRIDGSGQMA ITVDVEVASD TPHPARIGLN CQLAQVAERV NWLG LGPQE NYPDRLTAAC FDRWDLPLSD MYTPYVFPSE NGLRCGTREL NYGPHQWRGD FQFNI SRYS QQQLMETSHR HLLHAEEGTW LNIDGFHMGI GGDDSWSPSV SAEFQLSAGR YHYQLV WCQ K

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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

Concentration2.6 mg/mL
BufferpH: 7.9
Details: The lyophilized protein powder was reconstituted into a 5 mg/ml solution with 50 mM Tris-HCl (pH 7.9), 50 mM NaCl, 2 mM MgCl2 and 2mM Mercaptoethanol. 300 ul of this solution were loaded ...Details: The lyophilized protein powder was reconstituted into a 5 mg/ml solution with 50 mM Tris-HCl (pH 7.9), 50 mM NaCl, 2 mM MgCl2 and 2mM Mercaptoethanol. 300 ul of this solution were loaded onto a Superdex 200 10/300 GL (GE LifeSciences) size-exclusion chromatography column and eluted with 50 mM Tris-HCl (pH 7.9), 50 mM NaCl, 2 mM MgCl2 and 2 mM TCEP. Peak fractions corresponding to the tetrameric complex were pooled and concentrated to 2.6 mg/ml with a 50 kDa MWCO spin concentrator (Amicon, Millipore Co.). The sample were checked by SDS-PAGE with silver staining (Pierce silver staining kit, ThermoFischer Scientific).
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Quantifoil 400 mesh copper 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 ...Details: Quantifoil 400 mesh copper 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..
Detailsbeta-Galactosidase from E. coli was purchased from SIGMA-ALDRICH (G5635).

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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: 2.77 µm / Calibrated defocus min: 1.12 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 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 integrating mode 25 frames per movie
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 1389 / Average exposure time: 4.3 sec. / Average electron dose: 52.0 e/Å2
Details: images were collected in movie mode (25 frames in total)
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 307321
CTF correctionSoftware - Name: RELION
Initial angle assignmentType: OTHER
Details: the initial model was determined for a small sub-set using cistem
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: D2 (2x2 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 146475
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: 34

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