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Yorodumi- EMDB-1457: A test-bed for optimizing high-resolution single particle reconst... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1457 | |||||||||
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Title | A test-bed for optimizing high-resolution single particle reconstructions. | |||||||||
Map data | This is a single particle reconstruction of GroEL | |||||||||
Sample |
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Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 5.4 Å | |||||||||
Authors | Stagg SM / Lander GC / Quispe J / Voss NR / Cheng A / Bradlow H / Bradlow S / Carragher B / Potter CS | |||||||||
Citation | Journal: J Struct Biol / Year: 2008 Title: A test-bed for optimizing high-resolution single particle reconstructions. Authors: Scott M Stagg / Gabriel C Lander / Joel Quispe / Neil R Voss / Anchi Cheng / Henry Bradlow / Steven Bradlow / Bridget Carragher / Clinton S Potter / Abstract: It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of approximately 10A, but maps with resolutions of 5A or better are still ...It is becoming routine for cryoEM single particle reconstructions to result in 3D electron density maps with resolutions of approximately 10A, but maps with resolutions of 5A or better are still celebrated events. The electron microscope has a resolving power to better than 2A, and thus should not be a limiting factor; instead the practical limitations in resolution most likely arise from a combination of specimen preparation methods, data collection parameters, and data analysis procedures. With the aid of a highly automated system for acquiring images, coupled to a relational database to keep track of all processing parameters, we have taken a systematic approach to optimizing parameters affecting the resolution of single particle reconstructions. Using GroEL as a test-bed, we performed a series of 3D reconstructions where we systematically varied the number of particles used in computing the map, the accelerating voltage of the microscope, and the electron dose used to acquire the images. We also investigated methods for excluding unacceptable or "bad" particles from contributing to the final 3D map. Using relatively standard instrumentation (Tecnai F20, 4K x 4K CCD, side entry cold stage) and a completely automated approach, these approaches resulted in a map with a nominal resolution of 5.4A (FSC(0.5)) in which secondary structure is clearly discernable and the handedness of some of the alpha-helices in the GroEL structure can be determined. | |||||||||
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_1457.map.gz | 5.2 MB | EMDB map data format | |
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Header (meta data) | emd-1457-v30.xml emd-1457.xml | 9.7 KB 9.7 KB | Display Display | EMDB header |
Images | 1457.gif | 59.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1457 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1457 | HTTPS FTP |
-Validation report
Summary document | emd_1457_validation.pdf.gz | 255.6 KB | Display | EMDB validaton report |
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Full document | emd_1457_full_validation.pdf.gz | 254.7 KB | Display | |
Data in XML | emd_1457_validation.xml.gz | 5.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1457 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1457 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_1457.map.gz / Format: CCP4 / Size: 14.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | This is a single particle reconstruction of GroEL | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.63 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 : GroEL
Entire | Name: GroEL |
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Components |
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-Supramolecule #1000: GroEL
Supramolecule | Name: GroEL / type: sample / ID: 1000 / Oligomeric state: D7 14-mer / Number unique components: 1 |
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Molecular weight | Experimental: 800 KDa / Theoretical: 800 KDa |
-Macromolecule #1: GroEL
Macromolecule | Name: GroEL / type: protein_or_peptide / ID: 1 / Name.synonym: GroEL / Number of copies: 14 / Oligomeric state: homotetradecamer / Recombinant expression: Yes |
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Source (natural) | Organism: Escherichia coli (E. coli) / Cell: E. coli / Location in cell: cytosol |
Molecular weight | Experimental: 800 KDa / Theoretical: 800 KDa |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 4 mg/mL |
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Buffer | pH: 7.5 / Details: 100mM Hepes, 10mM Mg(OAc)2, 10mM KOAc, 2mM DTT |
Staining | Type: NEGATIVE / Details: not stained |
Grid | Details: Protochips C-flat grid: holey carbon with 2um holes and 2um spacing 400 mesh copper grid |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: OTHER Details: Vitrification instrument: Vitrobot. Grid plasma cleaned for 20s with Fischione 1020 plasma cleaner using 75% Argon 25% Oxygen mix. Method: Temperature of chamber was 4 degrees C. 0 seconds drain time. Single blot. 0 mm offset. 4 ul sample applied to grid. Blot for 3.5 seconds before plunging. |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Average: 102 K |
Alignment procedure | Legacy - Astigmatism: objective lens astigmatism was corrected automatically using Leginon |
Date | Jul 12, 2006 |
Image recording | Category: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 13 e/Å2 |
Tilt angle min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 100000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 100000 |
Sample stage | Specimen holder: Side entry liquid nitrogen-cooled cryo specimen holder Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
CTF correction | Details: Phase correction for each particle. |
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Final reconstruction | Applied symmetry - Point group: D7 (2x7 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 5.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN / Number images used: 55351 |
Final two d classification | Number classes: 1294 |