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Yorodumi- EMDB-0409: Single-particle cryo-EM reconstruction of the catalytic subunit o... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0409 | |||||||||
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Title | Single-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 using 200 keV | |||||||||
Map data | Single-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 (unsharpened) | |||||||||
Sample |
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Biological species | Mus musculus (house mouse) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.0 Å | |||||||||
Authors | Herzik Jr MA / Wu M / Lander GC | |||||||||
Citation | Journal: Nat Commun / Year: 2019 Title: High-resolution structure determination of sub-100 kDa complexes using conventional cryo-EM. Authors: Mark A Herzik / Mengyu Wu / Gabriel C Lander / Abstract: Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While ...Determining high-resolution structures of biological macromolecules amassing less than 100 kilodaltons (kDa) has been a longstanding goal of the cryo-electron microscopy (cryo-EM) community. While the Volta phase plate has enabled visualization of specimens in this size range, this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.7 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity to enable investigation of drug-target interactions. Our ~2.8 Å and ~3.2 Å structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the sub-nanometer cryo-EM structure of a sub-50 kDa protein. | |||||||||
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_0409.map.gz | 6 MB | EMDB map data format | |
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Header (meta data) | emd-0409-v30.xml emd-0409.xml | 21.2 KB 21.2 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_0409_fsc.xml | 5.4 KB | Display | FSC data file |
Images | emd_0409.png | 35.8 KB | ||
Others | emd_0409_additional.map.gz emd_0409_half_map_1.map.gz emd_0409_half_map_2.map.gz | 7.4 MB 6 MB 6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0409 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0409 | HTTPS FTP |
-Validation report
Summary document | emd_0409_validation.pdf.gz | 78.8 KB | Display | EMDB validaton report |
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Full document | emd_0409_full_validation.pdf.gz | 77.9 KB | Display | |
Data in XML | emd_0409_validation.xml.gz | 494 B | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0409 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-0409 | HTTPS FTP |
-Related structure data
Related structure data | 0406C 0407C 0408C 6nbbC 6nbcC 6nbdC C: citing same article (ref.) |
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Similar structure data | |
EM raw data | EMPIAR-10252 (Title: Catalytic subunit of protein kinase A bound to ATP, manganese, and IP20 movies obtained using Talos Arctica operating at 200 kV equipped with a K2 Data size: 5.5 TB Data #1: Raw, unaligned movie stacks of the catalytic subunit of protein kinase A bound to ATP, manganese, and IP20 acquired on a Talos Arctica using a K2 direct electron detector [micrographs - multiframe]) |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_0409.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Single-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 (unsharpened) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.1172 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Additional map: Single-particle cryo-EM reconstruction of the catalytic subunit of...
File | emd_0409_additional.map | ||||||||||||
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Annotation | Single-particle cryo-EM reconstruction of the catalytic subunit of protein kinase A bound to ATP and IP20 (sharpened) | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Odd half map
File | emd_0409_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_0409_half_map_2.map | ||||||||||||
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Annotation | Even half map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : catalytic subunit of mouse protein kinase A bound to ATP and IP20
Entire | Name: catalytic subunit of mouse protein kinase A bound to ATP and IP20 |
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Components |
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-Supramolecule #1: catalytic subunit of mouse protein kinase A bound to ATP and IP20
Supramolecule | Name: catalytic subunit of mouse protein kinase A bound to ATP and IP20 type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 43 KDa |
-Macromolecule #1: catalytic subunit of protein kinase A
Macromolecule | Name: catalytic subunit of protein kinase A / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: non-specific serine/threonine protein kinase |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: GNAAAAKKGS EQESVKEFLA KAKEDFLKKW ETPSQNTAQL DQFDRIKTLG TGSFGRVMLV KHKESGNHYA MKILDKQKVV KLKQIEHTL NEKRILQAVN FPFLVKLEFS FKDNSNLYMV MEYVAGGEMF SHLRRIGRFS EPHARFYAAQ IVLTFEYLHS L DLIYRDLK ...String: GNAAAAKKGS EQESVKEFLA KAKEDFLKKW ETPSQNTAQL DQFDRIKTLG TGSFGRVMLV KHKESGNHYA MKILDKQKVV KLKQIEHTL NEKRILQAVN FPFLVKLEFS FKDNSNLYMV MEYVAGGEMF SHLRRIGRFS EPHARFYAAQ IVLTFEYLHS L DLIYRDLK PENLLIDQQG YIQVTDFGFA KRVKGRTWTL CGTPEYLAPE IILSKGYNKA VDWWALGVLI YEMAAGYPPF FA DQPIQIY EKIVSGKVRF PSHFSSDLKD LLRNLLQVDL TKRFGNLKNG VNDIKNHKWF ATTDWIAIYQ RKVEAPFIPK FKG PGDTSN FDDYEEEEIR VSINEKCGKE FTEF |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 5 mg/mL | |||||||||||||||||||||
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Buffer | pH: 7 Component:
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Grid | Model: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER Details: Grids were plasma cleaned using a Solarus plasma cleaner (Gatan, Inc.) | |||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER Details: Sample was manually blotted for 4-5 seconds using Whatman No. 1 filter paper immediately prior to plunge-freezing.. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | #0 - Image recording ID: 1 / #0 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #0 - Detector mode: COUNTING / #0 - Number grids imaged: 2 / #0 - Number real images: 3176 / #0 - Average exposure time: 11.0 sec. / #0 - Average electron dose: 69.0 e/Å2 / #1 - Image recording ID: 2 / #1 - Film or detector model: GATAN K2 SUMMIT (4k x 4k) / #1 - Detector mode: COUNTING / #1 - Digitization - Dimensions - Width: 3710 pixel / #1 - Digitization - Dimensions - Height: 3838 pixel / #1 - Digitization - Sampling interval: 5.0 µm / #1 - Digitization - Frames/image: 1-44 / #1 - Number grids imaged: 2 / #1 - Number real images: 2402 / #1 - Average exposure time: 11.0 sec. / #1 - Average electron dose: 69.0 e/Å2 / #1 - Details: Specimen was tilted at 30 degrees |
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 FIELD / Cs: 2.7 mm / Nominal defocus max: 16.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 73000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |