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- EMDB-10533: Horse spleen apoferritin deposited with a microfluidic sprayer -

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Basic information

Entry
Database: EMDB / ID: EMD-10533
TitleHorse spleen apoferritin deposited with a microfluidic sprayer
Map dataFinal sharpened map
Sample
  • Complex: apoferritin from equine spleen
Function / homology
Function and homology information


: / intracellular sequestering of iron ion / ferric iron binding / ferrous iron binding / iron ion transport / iron ion binding / cytoplasm
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 light chain
Similarity search - Component
Biological speciesEquus caballus (horse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsKlebl DP / Muench SP
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/P026397/1 United Kingdom
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2020
Title: Sample deposition onto cryo-EM grids: from sprays to jets and back.
Authors: David P Klebl / Diana C F Monteiro / Dimitrios Kontziampasis / Florian Kopf / Frank Sobott / Howard D White / Martin Trebbin / Stephen P Muench /
Abstract: Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample ...Despite the great strides made in the field of single-particle cryogenic electron microscopy (cryo-EM) in microscope design, direct electron detectors and new processing suites, the area of sample preparation is still far from ideal. Traditionally, sample preparation involves blotting, which has been used to achieve high resolution, particularly for well behaved samples such as apoferritin. However, this approach is flawed since the blotting process can have adverse effects on some proteins and protein complexes, and the long blot time increases exposure to the damaging air-water interface. To overcome these problems, new blotless approaches have been designed for the direct deposition of the sample on the grid. Here, different methods of producing droplets for sample deposition are compared. Using gas dynamic virtual nozzles, small and high-velocity droplets were deposited on cryo-EM grids, which spread sufficiently for high-resolution cryo-EM imaging. For those wishing to pursue a similar approach, an overview is given of the current use of spray technology for cryo-EM grid preparation and areas for enhancement are pointed out. It is further shown how the broad aspects of sprayer design and operation conditions can be utilized to improve grid quality reproducibly.
History
DepositionDec 8, 2019-
Header (metadata) releaseApr 22, 2020-
Map releaseApr 22, 2020-
UpdateApr 22, 2020-
Current statusApr 22, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.036
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.036
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10533.png

Downloads & links

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Map

FileDownload / File: emd_10533.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal sharpened map
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.036 / Movie #1: 0.036
Minimum - Maximum-0.06200532 - 0.11503656
Average (Standard dev.)0.00069538626 (±0.007541221)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 214.00002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z214.000214.000214.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0620.1150.001

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Supplemental data

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Mask #1

Fileemd_10533_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_10533_additional.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap 1

Fileemd_10533_half_map_1.map
AnnotationHalfmap 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap 2

Fileemd_10533_half_map_2.map
AnnotationHalfmap 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 from equine spleen

EntireName: apoferritin from equine spleen
Components
  • Complex: apoferritin from equine spleen

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Supramolecule #1: apoferritin from equine spleen

SupramoleculeName: apoferritin from equine spleen / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Equus caballus (horse)

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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

Concentration9 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
30.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.01 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 295 K / Instrument: HOMEMADE PLUNGER
Details: sample sprayed onto grid using microfluidic nozzle and frozen 36 ms after spraying.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 690 / Average exposure time: 8.0 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 70425
CTF correctionSoftware - Name: Gctf
Final reconstructionApplied symmetry - Point group: O (octahedral) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 9633
Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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