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Yorodumi- EMDB-21023: Single-particle cryo-EM reconstruction of rabbit muscle aldolase ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-21023 | |||||||||
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Title | Single-particle cryo-EM reconstruction of rabbit muscle aldolase using 200 keV | |||||||||
Map data | Single-particle cryo-EM reconstruction of rabbit muscle aldolase | |||||||||
Sample |
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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 / glycolytic process / protein homotetramerization / positive regulation of cell migration Similarity search - Function | |||||||||
Biological species | Oryctolagus cuniculus (rabbit) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.13 Å | |||||||||
Authors | Wu M / Lander GC / Herzik MA | |||||||||
Funding support | United States, 1 items
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Citation | Journal: J Struct Biol X / Year: 2020 Title: Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV. Authors: Mengyu Wu / Gabriel C Lander / Mark A Herzik / Abstract: Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination ...Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as ∼1.7 Å resolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 kV electrons for high-resolution single-particle cryo-EM and applications such as structure-based drug design. | |||||||||
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_21023.map.gz | 59.4 MB | EMDB map data format | |
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Header (meta data) | emd-21023-v30.xml emd-21023.xml | 21.7 KB 21.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_21023_fsc.xml | 18.1 KB | Display | FSC data file |
Images | emd_21023.png | 231.9 KB | ||
Masks | emd_21023_msk_1.map | 512 MB | Mask map | |
Others | emd_21023_additional.map.gz emd_21023_half_map_1.map.gz emd_21023_half_map_2.map.gz | 409.7 MB 410.9 MB 411.1 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-21023 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-21023 | HTTPS FTP |
-Related structure data
Related structure data | 6v20MC 6v21C M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10338 (Title: Rabbit muscle aldolase movies obtained using a Talos Arctica (200 kV) equipped with a K2 Data size: 543.3 Data #1: Rabbit muscle aldolase movies obtained using a Talos Arctica (200 kV) equipped with a K2 [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_21023.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Single-particle cryo-EM reconstruction of rabbit muscle aldolase | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.562 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_21023_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Unsharpened map
File | emd_21023_additional.map | ||||||||||||
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Annotation | Unsharpened map | ||||||||||||
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Density Histograms |
-Half map: Odd half map
File | emd_21023_half_map_1.map | ||||||||||||
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Annotation | Odd half map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Even half map
File | emd_21023_half_map_2.map | ||||||||||||
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Annotation | Even half map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Aldolase from rabbit muscle
Entire | Name: Aldolase from rabbit muscleFructose-bisphosphate aldolase |
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Components |
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-Supramolecule #1: Aldolase from rabbit muscle
Supramolecule | Name: Aldolase from rabbit muscle / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 Details: Lyophilized rabbit muscle aldolase purchased from Sigma Aldrich was further purified to homogeneity. |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / Tissue: muscle |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 150 KDa |
-Macromolecule #1: Fructose-bisphosphate aldolase A
Macromolecule | Name: Fructose-bisphosphate aldolase A / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: fructose-bisphosphate aldolase |
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Source (natural) | Organism: Oryctolagus cuniculus (rabbit) / Tissue: muscle |
Molecular weight | Theoretical: 37.302602 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: HSHPALTPEQ KKELSDIAHR IVAPGKGILA ADESTGSIAK RLQSIGTENT EENRRFYRQL LLTADDRVNP CIGGVILFHE TLYQKADDG RPFPQVIKSK GGVVGIKVDK GVVPLAGTNG ETTTQGLDGL SERCAQYKKD GADFAKWRCV LKIGEHTPSA L AIMENANV ...String: HSHPALTPEQ KKELSDIAHR IVAPGKGILA ADESTGSIAK RLQSIGTENT EENRRFYRQL LLTADDRVNP CIGGVILFHE TLYQKADDG RPFPQVIKSK GGVVGIKVDK GVVPLAGTNG ETTTQGLDGL SERCAQYKKD GADFAKWRCV LKIGEHTPSA L AIMENANV LARYASICQQ NGIVPIVEPE ILPDGDHDLK RCQYVTEKVL AAVYKALSDH HIYLEGTLLK PNMVTPGHAC TQ KYSHEEI AMATVTALRR TVPPAVTGVT FLSGGQSEEE ASINLNAINK CPLLKPWALT FSYGRALQAS ALKAWGGKKE NLK AAQEEY VKRALANSLA CQGKYTP |
-Macromolecule #2: water
Macromolecule | Name: water / type: ligand / ID: 2 / Number of copies: 328 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.6 mg/mL | |||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Details: 15 Watts | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER Details: 3 uL of sample/grid was manually blotted for 4 seconds prior to immediate plunge-freezing in liquid nitrogen-cooled ethane.. | |||||||||
Details | Reconstituted from lyophilized form (Sigma Aldrich) |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 73000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-44 / Number grids imaged: 1 / Number real images: 3905 / Average exposure time: 11.0 sec. / Average electron dose: 67.0 e/Å2 Details: Images were collected using stage position navigation to target exposure. |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
-Image processing
-Atomic model buiding 1
Initial model | PDB ID: |
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Details | Starting model was generated by stripping PDB entry 5VY5 of all ligands and alternate conformations, then refining into the EM density using imposed symmetry while adjusting weighting/scoring according to estimated map resolution. The top 10 generated models (ranked based on quality metrics) were real-space refined using Phenix software. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT / Overall B value: 30 |
Output model | PDB-6v20: |