|Entry||Database: EMDB / ID: EMD-3407|
|Title||Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment|
|Keywords||Cryo-EM / Refinement of atomic models / assessment|
|Function / homology|
Function and homology information
GroEL-GroES complex / 'de novo' protein folding / chaperone cofactor-dependent protein refolding / virion assembly / protein refolding / response to radiation / unfolded protein binding / response to heat / protein folding / ATPase activity ...GroEL-GroES complex / 'de novo' protein folding / chaperone cofactor-dependent protein refolding / virion assembly / protein refolding / response to radiation / unfolded protein binding / response to heat / protein folding / ATPase activity / cell cycle / cell division / magnesium ion binding / membrane / ATP binding / identical protein binding / cytosol
GroEL-like apical domain superfamily / TCP-1-like chaperonin intermediate domain superfamily / GroEL-like equatorial domain superfamily / Chaperonin Cpn60, conserved site / Chaperonin Cpn60/TCP-1 family / Chaperonin Cpn60 / Chaperonin Cpn60
60 kDa chaperonin
|Biological species||Escherichia coli (E. coli)|
|Method||single particle reconstruction / cryo EM / negative staining / Resolution: 3.26 Å|
|Authors||Joseph AP / Malhotra S / Burnley T / Wood C / Clare DK / Winn M / Topf M|
|Citation||Journal: Methods / Year: 2016|
Title: Refinement of atomic models in high resolution EM reconstructions using Flex-EM and local assessment.
Authors: Agnel Praveen Joseph / Sony Malhotra / Tom Burnley / Chris Wood / Daniel K Clare / Martyn Winn / Maya Topf /
Abstract: As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high ...As the resolutions of Three Dimensional Electron Microscopic reconstructions of biological macromolecules are being improved, there is a need for better fitting and refinement methods at high resolutions and robust approaches for model assessment. Flex-EM/MODELLER has been used for flexible fitting of atomic models in intermediate-to-low resolution density maps of different biological systems. Here, we demonstrate the suitability of the method to successfully refine structures at higher resolutions (2.5-4.5Å) using both simulated and experimental data, including a newly processed map of Apo-GroEL. A hierarchical refinement protocol was adopted where the rigid body definitions are relaxed and atom displacement steps are reduced progressively at successive stages of refinement. For the assessment of local fit, we used the SMOC (segment-based Manders' overlap coefficient) score, while the model quality was checked using the Qmean score. Comparison of SMOC profiles at different stages of refinement helped in detecting regions that are poorly fitted. We also show how initial model errors can have significant impact on the goodness-of-fit. Finally, we discuss the implementation of Flex-EM in the CCP-EM software suite.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_3407.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.055 Å|
|Symmetry||Space group: 1|
CCP4 map header:
|Entire||Name: GroEL / Number of components: 1 / Oligomeric State: tetradecamer|
|Mass||Theoretical: 56 kDa / Experimental: 56 kDa|
-Component #1: protein, GroEL
|Protein||Name: GroEL / a.k.a: 60 kDa Chaperonin / Oligomeric Details: tetradecamer / Recombinant expression: No|
|Mass||Theoretical: 800 kDa / Experimental: 800 kDa|
|Source||Species: Escherichia coli (E. coli)|
|Source (natural)||Location in cell: cytoplasm|
|External references||InterPro: Chaperonin Cpn60 / UniProt: 60 kDa chaperonin / Gene Ontology: GroEL-GroES complex|
|Specimen||Specimen state: Particle / Method: negative staining, cryo EM|
|Sample solution||Specimen conc.: 4 mg/mL / Buffer solution: 50 mM Tris-HCl, 50 mM KCl and 10 mM MgCl2 / pH: 7.4|
|Support film||400 mesh C-flat r2/2|
|Staining||3.5ul of protein was added to glow discharged C-flat r2/2 grid which were then blotted and plunge frozen|
|Vitrification||Instrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Temperature: 100 K / Humidity: 40 %|
Method: 3.5ul of protein was added to glow discharged C-flat r2/2 grid which were then blotted and plunge frozen
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS / Date: Oct 9, 2015|
Details: The images were recorded using a dose rate of ~8 electrons/A2/s (~9 electrons/pixel/s) with 20 movie frames collected over a 4 second exposure
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 32 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 130000 X (nominal), 47400 X (calibrated)|
Astigmatism: Objective lens astigmatism was corrected at the magnification used to collect the data
Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000 - 2800 nm / Energy filter: Gatan / Energy window: 0-20 eV
|Specimen Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 90 (90 - 91 K)|
|Camera||Detector: GATAN K2 QUANTUM (4k x 4k)|
|Image acquisition||Number of digital images: 370 / Sampling size: 5 µm / Bit depth: 32 |
Details: Each image is summation of 20 frames recorded over 4 seconds
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