|Entry||Database: EMDB / ID: EMD-1457|
|Title||A test-bed for optimizing high-resolution single particle reconstructions.|
|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.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_1457.map.gz / Format: CCP4 / Size: 14.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.63 Å|
|Symmetry||Space group: 1|
CCP4 map header:
|Entire||Name: GroEL / Oligomeric State: D7 14-mer / Number of components: 1|
|Mass||Theoretical: 800 kDa / Experimental: 800 kDa|
-Component #1: protein, GroEL
|Protein||Name: GroEL / a.k.a: GroEL / Oligomeric Details: homotetradecamer / Recombinant expression: Yes / Number of Copies: 14|
|Mass||Theoretical: 800 kDa / Experimental: 800 kDa|
|Source||Species: Escherichia coli (E. coli)|
|Source (natural)||Location in cell: cytosol / Cell: E. coli|
|Specimen||Specimen state: Particle / Method: negative staining, cryo EM|
|Sample solution||Specimen conc.: 4 mg/mL|
Buffer solution: 100mM Hepes, 10mM Mg(OAc)2, 10mM KOAc, 2mM DTT
|Support film||Protochips C-flat grid: holey carbon with 2um holes and 2um spacing 400 mesh copper grid|
|Vitrification||Instrument: FEI VITROBOT / Cryogen name: ETHANE / Temperature: 93 K / Humidity: 100 %|
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.
Details: Vitrification instrument: Vitrobot. Grid plasma cleaned for 20s with Fischione 1020 plasma cleaner using 75% Argon 25% Oxygen mix.
-Electron microscopy imaging
Model: Tecnai F20 / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI F20 / Date: Jul 12, 2006|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 120 kV / Electron dose: 13 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 100000 X (nominal), 100000 X (calibrated)|
Astigmatism: objective lens astigmatism was corrected automatically using Leginon
Cs: 2 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 3000 nm
|Specimen Holder||Holder: Side entry liquid nitrogen-cooled cryo specimen holder|
Model: GATAN LIQUID NITROGEN / Temperature: 102
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
|Processing||Method: single particle reconstruction / Number of class averages: 1294 / Number of projections: 55351 / Applied symmetry: D7 (2x7 fold dihedral)|
|3D reconstruction||Algorithm: single particle reconstruction / Software: EMAN / CTF correction: Phase correction for each particle. / Resolution: 5.4 Å / Resolution method: FSC 0.5|
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