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- EMDB-3645: CryoEM density of TcdA1 in prepore state (SPHIRE tutorial) -

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Basic information

Entry
Database: EMDB / ID: EMD-3645
TitleCryoEM density of TcdA1 in prepore state (SPHIRE tutorial)
Map dataCryoEM density of TcdA1 in prepore state
Sample
  • Complex: TcdA1
Biological speciesPhotorhabdus luminescens (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsRaunser S / Gatsogiannis S / Roderer S
CitationJournal: J Vis Exp / Year: 2017
Title: High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE.
Authors: Toshio Moriya / Michael Saur / Markus Stabrin / Felipe Merino / Horatiu Voicu / Zhong Huang / Pawel A Penczek / Stefan Raunser / Christos Gatsogiannis /
Abstract: SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) ...SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 Å structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.
History
DepositionMar 21, 2017-
Header (metadata) releaseApr 5, 2017-
Map releaseMay 24, 2017-
UpdateMay 2, 2018-
Current statusMay 2, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.055
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3645.png

Downloads & links

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Map

FileDownload / File: emd_3645.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryoEM density of TcdA1 in prepore state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 352 pix.
= 401.28 Å		1.14 Å/pix.
x 352 pix.
= 401.28 Å		1.14 Å/pix.
x 352 pix.
= 401.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.14 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.12000273 - 0.2384355
Average (Standard dev.)0.0010246624 (±0.008890688)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-176-176-176
Dimensions352352352
Spacing352352352
CellA=B=C: 401.28 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z401.280401.280401.280
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-176-176-176
NC/NR/NS352352352
D min/max/mean-0.1200.2380.001

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Supplemental data

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Sample components

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Entire : TcdA1

EntireName: TcdA1
Components
  • Complex: TcdA1

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Supramolecule #1: TcdA1

SupramoleculeName: TcdA1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Prepore state
Source (natural)Organism: Photorhabdus luminescens (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pET-19b
Molecular weightTheoretical: 1.4 MDa

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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: 5
Component:
ConcentrationName
20.0 mMTRIS
150.0 mMNaCl
0.05 %Tween-20
GridModel: Quantifoil 2/1 2nm C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: GATAN CRYOPLUNGE 3

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DetailsCs corrected microscope
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Number real images: 112 / Average electron dose: 2.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal magnification: 59000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 8416
CTF correctionSoftware - Name: SPHIRE
Startup modelType of model: OTHER
Details: initial model was obtained using VIPER (SPHIRE) from 2D class averages
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPHIRE
Details: The density was filtered to its local resolution, using localfilt (SPHIRE)
Number images used: 4943
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SPHIRE
Final angle assignmentType: PROJECTION MATCHING / Software - Name: SPHIRE
Final 3D classificationNumber classes: 2 / Avg.num./class: 4500 / Software - Name: SPHIRE

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