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Yorodumi- PDB-8ehg: Rabbit muscle aldolase determined using single-particle cryo-EM w... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8ehg | ||||||||||||
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Title | Rabbit muscle aldolase determined using single-particle cryo-EM with Apollo camera. | ||||||||||||
Components | Fructose-bisphosphate aldolase A | ||||||||||||
Keywords | SUGAR BINDING PROTEIN / glycolysis | ||||||||||||
Function / homology | Function and homology information 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 | Oryctolagus cuniculus (rabbit) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.24 Å | ||||||||||||
Authors | Peng, R. / Fu, X. / Mendez, J.H. / Randolph, P.H. / Bammes, B. / Stagg, S.M. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: J Struct Biol X / Year: 2023 Title: Characterizing the resolution and throughput of the Apollo direct electron detector. Authors: Ruizhi Peng / Xiaofeng Fu / Joshua H Mendez / Peter S Randolph / Benjamin E Bammes / Scott M Stagg / Abstract: Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been ...Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 Å resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 Å resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 Å resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8ehg.cif.gz | 312.6 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8ehg.ent.gz | 203.6 KB | Display | PDB format |
PDBx/mmJSON format | 8ehg.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8ehg_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
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Full document | 8ehg_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 8ehg_validation.xml.gz | 57.2 KB | Display | |
Data in CIF | 8ehg_validation.cif.gz | 86 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/eh/8ehg ftp://data.pdbj.org/pub/pdb/validation_reports/eh/8ehg | HTTPS FTP |
-Related structure data
Related structure data | 28147MC 8emqC 8en7C M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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Noncrystallographic symmetry (NCS) | NCS domain:
NCS domain segments:
NCS oper:
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-Components
#1: Protein | Mass: 39394.875 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: muscle / References: UniProt: P00883, fructose-bisphosphate aldolase |
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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 |
-Sample preparation
Component | Name: Aldolase from rabbit muscle / Type: COMPLEX Details: Plays a key role in glycolysis and gluconeogenesis. In addition, may also function as scaffolding protein. Entity ID: all / Source: NATURAL | ||||||||||||||||||||
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Molecular weight | Value: 14921 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / Tissue: muscle | ||||||||||||||||||||
Buffer solution | pH: 7.5 / Details: DTT are added freshly before use. | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
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 |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 72621 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 80 µm / Alignment procedure: ZEMLIN TABLEAU |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 1.347 sec. / Electron dose: 60 e/Å2 / Film or detector model: OTHER / Num. of grids imaged: 1 / Num. of real images: 8682 Details: Images were collected using Direct Electron Apollo camera at a fixed movie frame rate of 60 frames/second. |
Image scans | Width: 8192 / Height: 8192 |
-Processing
Software |
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EM software |
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Image processing | Details: Direct Electron Apollo | ||||||||||||||||||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 3805094 Details: particles were picked using templates projected from EMD-21023. | ||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: D2 (2x2 fold dihedral) | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 722778 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||||||||||||||||||
Displacement parameters | Biso mean: 8.14 Å2 | ||||||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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Refine LS restraints NCS |
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