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- EMDB-8614: 3D-reconstruction of O3-33 from a grid prepared with multiple rou... -

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Basic information

Entry
Database: EMDB / ID: EMD-8614
Title3D-reconstruction of O3-33 from a grid prepared with multiple rounds of blotting
Map data3D-reconstruction of O3-33
Sample
  • Complex: O3-33
    • Protein or peptide: Computationally designed octahedral protein cage O3-33
Function / homology
Function and homology information


propanediol degradation polyhedral organelle / propanediol catabolic process / 4 iron, 4 sulfur cluster binding / metal ion binding
Similarity search - Function
Bacterial microcompartment shell protein PduT / CcmK/CsoS1, bacterial microcompartment domain / : / Bacterial microcompartment (BMC) domain profile. / BMC domain / Bacterial microcompartment domain / BMC / CcmK-like superfamily
Similarity search - Domain/homology
: / Bacterial microcompartment shell protein PduT
Similarity search - Component
Biological speciesunidentified (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.3 Å
AuthorsSnijder J / Borst AJ / Dosey A / Walls AC / Burrell A / Reddy V / Kollman J / Veesler D
Funding support United States, Netherlands, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM120553-01 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI070771 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM118396-01 United States
Netherlands Organization for Scientific ResearchNWO, Rubicon 019.2015.2.310.006 Netherlands
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32GM008268 United States
EMBOALTF 933-2015 United States
CitationJournal: J Struct Biol / Year: 2017
Title: Vitrification after multiple rounds of sample application and blotting improves particle density on cryo-electron microscopy grids.
Authors: Joost Snijder / Andrew J Borst / Annie Dosey / Alexandra C Walls / Anika Burrell / Vijay S Reddy / Justin M Kollman / David Veesler /
Abstract: Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain ...Single particle cryo-electron microscopy (cryoEM) is becoming widely adopted as a tool for structural characterization of biomolecules at near-atomic resolution. Vitrification of the sample to obtain a dense distribution of particles within a single field of view remains a major bottleneck for the success of such experiments. Here, we describe a simple and cost-effective method to increase the density of frozen-hydrated particles on grids with holey carbon support films. It relies on performing multiple rounds of sample application and blotting prior to plunge freezing in liquid ethane. We show that this approach is generally applicable and significantly increases particle density for a range of samples, such as small protein complexes, viruses and filamentous assemblies. The method is versatile, easy to implement, minimizes sample requirements and can enable characterization of samples that would otherwise resist structural studies using single particle cryoEM.
History
DepositionFeb 23, 2017-
Header (metadata) releaseMar 15, 2017-
Map releaseMar 15, 2017-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.12
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8614.png

Downloads & links

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Map

FileDownload / File: emd_8614.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D-reconstruction of O3-33
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.5 Å/pix.
x 128 pix.
= 320. Å		2.5 Å/pix.
x 128 pix.
= 320. Å		2.5 Å/pix.
x 128 pix.
= 320. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.035881292 - 0.2295262
Average (Standard dev.)0.0024040714 (±0.017552491)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 320.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z320.000320.000320.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0360.2300.002

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Supplemental data

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Sample components

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Entire : O3-33

EntireName: O3-33
Components
  • Complex: O3-33
    • Protein or peptide: Computationally designed octahedral protein cage O3-33

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Supramolecule #1: O3-33

SupramoleculeName: O3-33 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: unidentified (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 480 KDa

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Macromolecule #1: Computationally designed octahedral protein cage O3-33

MacromoleculeName: Computationally designed octahedral protein cage O3-33
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: unidentified (others)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MSQAIGILEL TSIAAGMELG DAMLKSANVD LLVSKTISPG KFLLMLGGDI GAIQQAIETG TSQAGELLVD SLVLANIHPS VLPAISGLNS VDKRQAVGIV ETWSVAACIS AADRAVKGSN VTLVRVHMAF GIGGKCYMVV AGDVSDVALA VTVASSSAGA YGLLVYASLI PRPHEAMWRQ MVEGlehhhh hh

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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

Concentration0.1 mg/mL
BufferpH: 8 / Component:
ConcentrationName
25.0 mMTris
150.0 mMNaCl
GridModel: C-flat / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 100 / Average exposure time: 10.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Calibrated magnification: 40000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf/Relion
Startup modelType of model: OTHER / Details: ab initio e2initialmodel.py
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 6000
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final 3D classificationSoftware - Name: RELION (ver. 2.0)

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