+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3645 | |||||||||
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Title | CryoEM density of TcdA1 in prepore state (SPHIRE tutorial) | |||||||||
Map data | CryoEM density of TcdA1 in prepore state | |||||||||
Sample |
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Biological species | Photorhabdus luminescens (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.5 Å | |||||||||
Authors | Raunser S / Gatsogiannis S / Roderer S | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3645.map.gz | 6.4 MB | EMDB map data format | |
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Header (meta data) | emd-3645-v30.xml emd-3645.xml | 11.5 KB 11.5 KB | Display Display | EMDB header |
Images | emd_3645.png | 90 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3645 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3645 | HTTPS FTP |
-Related structure data
Related structure data | |
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Similar structure data | |
EM raw data | EMPIAR-10089 (Title: Single Particle cryoEM Dataset of TcdA1 in prepore state (SPHIRE tutorial) Data size: 72.8 Data #1: Unaligned multi-frame micrographs of TcdA1 in prepore state (SPHIRE tutorial dataset) [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_3645.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | CryoEM density of TcdA1 in prepore state | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.14 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : TcdA1
Entire | Name: TcdA1 |
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Components |
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-Supramolecule #1: TcdA1
Supramolecule | Name: TcdA1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: Prepore state |
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Source (natural) | Organism: Photorhabdus luminescens (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant plasmid: pET-19b |
Molecular weight | Theoretical: 1.4 MDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 5 Component:
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Grid | Model: Quantifoil 2/1 2nm C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE | ||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Instrument: GATAN CRYOPLUNGE 3 |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Details | Cs corrected microscope |
Image recording | Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 112 / Average electron dose: 2.5 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Particle selection | Number selected: 8416 |
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CTF correction | Software - Name: SPHIRE |
Startup model | Type of model: OTHER Details: initial model was obtained using VIPER (SPHIRE) from 2D class averages |
Initial angle assignment | Type: PROJECTION MATCHING / Software - Name: SPHIRE |
Final 3D classification | Number classes: 2 / Avg.num./class: 4500 / Software - Name: SPHIRE |
Final angle assignment | Type: PROJECTION MATCHING / Software - Name: SPHIRE |
Final reconstruction | Applied 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 |