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- EMDB-30685: Krios G4 apoferritin test with K3/BioQuantum SerialEM BIS 1x1x4 -

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Basic information

Entry
Database: EMDB / ID: EMD-30685
TitleKrios G4 apoferritin test with K3/BioQuantum SerialEM BIS 1x1x4
Map dataSharpened apoferritin map
Sample
  • Complex: apoferritin
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / Neutrophil degranulation / endocytic vesicle lumen / ferric iron binding / autophagosome ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / Neutrophil degranulation / endocytic vesicle lumen / ferric iron binding / autophagosome / iron ion transport / intracellular iron ion homeostasis / immune response / iron ion binding / negative regulation of cell population proliferation / mitochondrion / extracellular region / identical protein binding / membrane / cytosol
Similarity search - Function
Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily
Similarity search - Domain/homology
Ferritin heavy chain
Similarity search - Component
Biological speciesmus musc (Temminck's mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.35 Å
AuthorsDanev R
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Science and Technology18069571 Japan
CitationJournal: Microscopy (Oxf) / Year: 2021
Title: Cryo-EM performance testing of hardware and data acquisition strategies.
Authors: Radostin Danev / Haruaki Yanagisawa / Masahide Kikkawa /
Abstract: The increasing popularity and adoption rate of cryo-electron microscopy (cryo-EM) is evidenced by a growing number of new microscope installations around the world. The quality and reliability of the ...The increasing popularity and adoption rate of cryo-electron microscopy (cryo-EM) is evidenced by a growing number of new microscope installations around the world. The quality and reliability of the instruments improved dramatically in recent years, but site-specific issues or unnoticed problems during installation could undermine productivity. Newcomers to the field may also have limited experience and/or low confidence in the capabilities of the equipment or their own skills. Therefore, it is recommended to perform an initial test of the complete cryo-EM workflow with an 'easy' test sample, such as apoferritin, before starting work with real and challenging samples. Analogous test experiments are also recommended for the quantification of new data acquisition approaches or imaging hardware. Here, we present the results from our initial tests of a recently installed Krios G4 electron microscope equipped with two latest generation direct electron detector cameras-Gatan K3 and Falcon 4. Three beam-image shift-based data acquisition strategies were also tested. We detail the methodology and discuss the critical parameters and steps for performance testing. The two cameras performed equally, and the single- and multi-shot per-hole acquisition schemes produced comparable results. We also evaluated the effects of environmental factors and optical flaws on data quality. Our results reaffirmed the exceptional performance of the software aberration correction in Relion in dealing with severe coma aberration. We hope that this work will help cryo-EM teams in their testing and troubleshooting of hardware and data collection approaches.
History
DepositionNov 18, 2020-
Header (metadata) releaseDec 2, 2020-
Map releaseDec 2, 2020-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_30685.map.gz / Format: CCP4 / Size: 476.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened apoferritin map
Voxel sizeX=Y=Z: 0.599 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.21423328 - 0.7499473
Average (Standard dev.)-1.581796e-06 (±0.012655616)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions500500500
Spacing500500500
CellA=B=C: 299.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5990.5990.599
M x/y/z500500500
origin x/y/z0.0000.0000.000
length x/y/z299.500299.500299.500
α/β/γ90.00090.00090.000
start NX/NY/NZ139118109
NX/NY/NZ123164187
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS500500500
D min/max/mean-0.2140.750-0.000

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Supplemental data

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Sample components

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Entire : apoferritin

EntireName: apoferritin
Components
  • Complex: apoferritin

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Supramolecule #1: apoferritin

SupramoleculeName: apoferritin / type: complex / ID: 1 / Parent: 0 / Details: mouse heavy chain
Source (natural)Organism: mus musc (Temminck's mouse)
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Molecular weightTheoretical: 500 KDa

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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

Concentration12 mg/mL
BufferpH: 7.5 / Details: HEPES-NaOH
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa / Details: Harrick Plasma Cleaner on HIGH
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 77.0 K
Alignment procedureComa free - Residual tilt: 0.1 mrad
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 15 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.0 µm / Number grids imaged: 1 / Number real images: 3472 / Average exposure time: 3.04 sec. / Average electron dose: 68.7 e/Å2
Details: Beam-image shift acquisition with a custom SerialEM script. Used SerialEM beam-tilt compensated image shift. Acquisition pattern: 1x1 holes, 4 images/hole
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 0.8 µm / Calibrated defocus min: 0.2 µm / Calibrated magnification: 125000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.8 µm / Nominal defocus min: 0.2 µm / Nominal magnification: 215000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 306000
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER / Details: previous reconstruction
Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 1.35 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 203
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.1)

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