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- EMDB-30106: 3.9A apoferritin from JEM-2200FS + DE-20 at 80K, RELION 3.0 proce... -

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Basic information

Entry
Database: EMDB / ID: EMD-30106
Title3.9A apoferritin from JEM-2200FS + DE-20 at 80K, RELION 3.0 processing, manual acquisition.
Map data3.9A apoferritin from JEM-2200FS DE-20 at 80K, RELION 3.0 processing, manual acquisition, postprocessed map.
Sample
  • Complex: Apoferritin, 3.9A, JEM-2200FS + DE-20, 80K, RELION 3.0Ferritin
Function / homology
Function and homology information


ferric iron binding / iron ion transport / intracellular iron ion homeostasis
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
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsBurton-Smith RN / Kayama Y / Song C / Kato T / Murata K
CitationJournal: Sci Rep / Year: 2021
Title: Below 3 Å structure of apoferritin using a multipurpose TEM with a side entry cryoholder.
Authors: Yoko Kayama / Raymond N Burton-Smith / Chihong Song / Naoya Terahara / Takayuki Kato / Kazuyoshi Murata /
Abstract: Recently, the structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method. Many results of cryo-EM SPA ...Recently, the structural analysis of protein complexes by cryo-electron microscopy (cryo-EM) single particle analysis (SPA) has had great impact as a biophysical method. Many results of cryo-EM SPA are based on data acquired on state-of-the-art cryo-electron microscopes customized for SPA. These are currently only available in limited locations around the world, where securing machine time is highly competitive. One potential solution for this time-competitive situation is to reuse existing multi-purpose equipment, although this comes with performance limitations. Here, a multi-purpose TEM with a side entry cryo-holder was used to evaluate the potential of high-resolution SPA, resulting in a 3 Å resolution map of apoferritin with local resolution extending to 2.6 Å. This map clearly showed two positions of an aromatic side chain. Further, examination of optimal imaging conditions depending on two different multi-purpose electron microscope and camera combinations was carried out, demonstrating that higher magnifications are not always necessary or desirable. Since automation is effectively a requirement for large-scale data collection, and augmenting the multi-purpose equipment is possible, we expanded testing by acquiring data with SerialEM using a β-galactosidase test sample. This study demonstrates the possibilities of more widely available and established electron microscopes, and their applications for cryo-EM SPA.
History
DepositionMar 12, 2020-
Header (metadata) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMar 17, 2021-
Current statusMar 17, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_30106.png

Downloads & links

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Map

FileDownload / File: emd_30106.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3.9A apoferritin from JEM-2200FS DE-20 at 80K, RELION 3.0 processing, manual acquisition, postprocessed map.
Voxel sizeX=Y=Z: 0.71 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.05670285 - 0.08440076
Average (Standard dev.)5.43113e-06 (±0.004043384)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 284.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.710.710.71
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z284.000284.000284.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0570.0840.000

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Supplemental data

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Mask #1

Fileemd_30106_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3.9A apoferritin from JEM-2200FS DE-20 at 80K, RELION...

Fileemd_30106_half_map_1.map
Annotation3.9A apoferritin from JEM-2200FS DE-20 at 80K, RELION 3.0 processing, manual acquisition, half map 1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: 3.9A apoferritin from JEM-2200FS DE-20 at 80K, RELION...

Fileemd_30106_half_map_2.map
Annotation3.9A apoferritin from JEM-2200FS DE-20 at 80K, RELION 3.0 processing, manual acquisition, half map 2.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Apoferritin, 3.9A, JEM-2200FS + DE-20, 80K, RELION 3.0

EntireName: Apoferritin, 3.9A, JEM-2200FS + DE-20, 80K, RELION 3.0Ferritin
Components
  • Complex: Apoferritin, 3.9A, JEM-2200FS + DE-20, 80K, RELION 3.0Ferritin

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Supramolecule #1: Apoferritin, 3.9A, JEM-2200FS + DE-20, 80K, RELION 3.0

SupramoleculeName: Apoferritin, 3.9A, JEM-2200FS + DE-20, 80K, RELION 3.0
type: complex / ID: 1 / Parent: 0 / Details: Octahedral symmetry.
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
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

BufferpH: 7
GridModel: Quantifoil R1.2/1.3 / Material: MOLYBDENUM / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL 2200FS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 4.2 mm / Nominal magnification: 80000
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 15 eV
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Average exposure time: 5.0 sec. / Average electron dose: 50.0 e/Å2

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1.10)
Startup modelType of model: OTHER / Details: Ab initio model generated by cisTEM.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 23056
FSC plot (resolution estimation)

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