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Open data
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Basic information
Entry | Database: PDB / ID: 7k9l | |||||||||
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Title | Aldolase, rabbit muscle (no beam-tilt refinement) | |||||||||
![]() | Fructose-bisphosphate aldolase A | |||||||||
![]() | LYASE / glycolysis / gluconeogenesis / Carbohydrate degradation / Homotetramer | |||||||||
Function / homology | ![]() negative regulation of Arp2/3 complex-mediated actin nucleation / fructose-bisphosphate aldolase / fructose-bisphosphate aldolase activity / M band / I band / fructose 1,6-bisphosphate metabolic process / glycolytic process / protein homotetramerization / positive regulation of cell migration / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å | |||||||||
![]() | Cianfrocco, M.A. / Kearns, S.E. / Cash, J.N. / Li, Y. | |||||||||
Funding support | ![]()
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![]() | ![]() Title: High-resolution cryo-EM using beam-image shift at 200 keV. Authors: Jennifer N Cash / Sarah Kearns / Yilai Li / Michael A Cianfrocco / ![]() Abstract: Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it ...Recent advances in single-particle cryo-electron microscopy (cryo-EM) data collection utilize beam-image shift to improve throughput. Despite implementation on 300 keV cryo-EM instruments, it remains unknown how well beam-image-shift data collection affects data quality on 200 keV instruments and the extent to which aberrations can be computationally corrected. To test this, a cryo-EM data set for aldolase was collected at 200 keV using beam-image shift and analyzed. This analysis shows that the instrument beam tilt and particle motion initially limited the resolution to 4.9 Å. After particle polishing and iterative rounds of aberration correction in , a 2.8 Å resolution structure could be obtained. This analysis demonstrates that software correction of microscope aberrations can provide a significant improvement in resolution at 200 keV. | |||||||||
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 231.8 KB | Display | ![]() |
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PDB format | ![]() | 189.7 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 22754MC ![]() 7k9xC ![]() 7ka2C ![]() 7ka3C ![]() 7ka4C M: map data used to model this data C: citing same article ( |
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Similar structure data | |
EM raw data | ![]() Data size: 87.6 Data #1: Unaligned micrographs of aldolase collected with beam-tilt at 200 kV [micrographs - single frame]) |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 39263.672 Da / Num. of mol.: 4 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Homotetramer of aldolase / Type: COMPLEX / Entity ID: all / Source: NATURAL | ||||||||||||
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Molecular weight | Value: 157 kDa/nm / Experimental value: YES | ||||||||||||
Source (natural) | Organism: ![]() ![]() | ||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||
Buffer component |
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Specimen | Conc.: 1.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: Pure aldolase isolated from rabbit muscle was purchased as a lyophilized powder (Sigma Aldrich) and solubilized in 20 mM HEPES (pH 7.5), 50 mM NaCl at 1.6 mg/ml. Sample was blotted for 4 ...Details: Pure aldolase isolated from rabbit muscle was purchased as a lyophilized powder (Sigma Aldrich) and solubilized in 20 mM HEPES (pH 7.5), 50 mM NaCl at 1.6 mg/ml. Sample was blotted for 4 seconds with Whatman No. #1 filter paper immediately prior to plunge freezing in liquid ethane cooled by liquid nitrogen. | ||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 45000 X / Nominal defocus max: 2 nm / Nominal defocus min: 0.8 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm |
Image recording | Average exposure time: 10 sec. / Electron dose: 42 e/Å2 / Film or detector model: GATAN K2 BASE (4k x 4k) Details: Images were collected on a Talos Arctica transmission electron microscope (Thermo Fisher) operating at 200 keV with a gun lens of 6, a spot size of 6, 70 um C2 aperture and 100 um objective ...Details: Images were collected on a Talos Arctica transmission electron microscope (Thermo Fisher) operating at 200 keV with a gun lens of 6, a spot size of 6, 70 um C2 aperture and 100 um objective aperture using beam-image shift. Movies were collected using a K2 direct electron detector (Gatan Inc.) operating in counting mode at 45,000x corresponding to a physical pixel size of 0.91 A/pixel with a 10 sec exposure using 200 ms per frame. Using an exposure rate of 4.204 e/pix/sec, each movie had a total dose of approximately 42 e/A2 for the 2,111 movies over a defocus 0.8-2 um. |
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Processing
Software | Name: PHENIX / Version: 1.14_3260: / Classification: refinement | ||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||
Particle selection | Num. of particles selected: 718578 | ||||||||||||
3D reconstruction | Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 186471 / Symmetry type: POINT |