+Open data
-Basic information
Entry | Database: PDB / ID: 8vsu | ||||||
---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of LKB1-STRADalpha-MO25alpha heterocomplex | ||||||
Components |
| ||||||
Keywords | TRANSFERASE / serine/threonine kinase / pseudokinase / complex | ||||||
Function / homology | Function and homology information positive regulation of vesicle transport along microtubule / intracellular protein-containing complex / LRR domain binding / Golgi localization / negative regulation of potassium ion transmembrane transporter activity / AMPK inhibits chREBP transcriptional activation activity / negative regulation of potassium ion transmembrane transport / dendrite extension / negative regulation of epithelial cell proliferation involved in prostate gland development / serine/threonine protein kinase complex ...positive regulation of vesicle transport along microtubule / intracellular protein-containing complex / LRR domain binding / Golgi localization / negative regulation of potassium ion transmembrane transporter activity / AMPK inhibits chREBP transcriptional activation activity / negative regulation of potassium ion transmembrane transport / dendrite extension / negative regulation of epithelial cell proliferation involved in prostate gland development / serine/threonine protein kinase complex / cellular hypotonic response / tissue homeostasis / response to thyroid hormone / epithelial cell proliferation involved in prostate gland development / Energy dependent regulation of mTOR by LKB1-AMPK / positive thymic T cell selection / vasculature development / regulation of Wnt signaling pathway / anoikis / negative regulation of cold-induced thermogenesis / response to glucagon / FOXO-mediated transcription of cell death genes / regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / positive regulation of axonogenesis / response to ionizing radiation / cellular response to UV-B / establishment of cell polarity / regulation of dendrite morphogenesis / response to lipid / activation of protein kinase activity / G1 to G0 transition / protein kinase activator activity / positive regulation of transforming growth factor beta receptor signaling pathway / intrinsic apoptotic signaling pathway by p53 class mediator / protein localization to nucleus / positive regulation of autophagy / positive regulation of gluconeogenesis / negative regulation of TORC1 signaling / protein export from nucleus / protein dephosphorylation / protein serine/threonine kinase activator activity / axonogenesis / positive regulation of peptidyl-threonine phosphorylation / regulation of signal transduction by p53 class mediator / response to activity / regulation of cell growth / peptidyl-threonine phosphorylation / negative regulation of canonical Wnt signaling pathway / positive regulation of protein serine/threonine kinase activity / negative regulation of cell growth / kinase binding / Z disc / autophagy / positive regulation of protein localization to nucleus / p53 binding / glucose homeostasis / T cell receptor signaling pathway / spermatogenesis / peptidyl-serine phosphorylation / secretory granule lumen / Regulation of TP53 Activity through Phosphorylation / ficolin-1-rich granule lumen / protein autophosphorylation / regulation of cell cycle / non-specific serine/threonine protein kinase / intracellular signal transduction / protein phosphorylation / negative regulation of cell population proliferation / protein serine kinase activity / protein serine/threonine kinase activity / DNA damage response / Neutrophil degranulation / protein-containing complex binding / magnesium ion binding / signal transduction / mitochondrion / extracellular exosome / extracellular region / nucleoplasm / ATP binding / membrane / nucleus / cytosol / cytoplasm Similarity search - Function | ||||||
Biological species | Homo sapiens (human) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å | ||||||
Authors | Chan, L.M. / Courteau, B.J. / Verba, K.A. | ||||||
Funding support | 1items
| ||||||
Citation | Journal: J Struct Biol / Year: 2024 Title: High-resolution single-particle imaging at 100-200 keV with the Gatan Alpine direct electron detector. Authors: Lieza M Chan / Brandon J Courteau / Allison Maker / Mengyu Wu / Benjamin Basanta / Hev Mehmood / David Bulkley / David Joyce / Brian C Lee / Stephen Mick / Cory Czarnik / Sahil Gulati / ...Authors: Lieza M Chan / Brandon J Courteau / Allison Maker / Mengyu Wu / Benjamin Basanta / Hev Mehmood / David Bulkley / David Joyce / Brian C Lee / Stephen Mick / Cory Czarnik / Sahil Gulati / Gabriel C Lander / Kliment A Verba / Abstract: Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments ...Developments in direct electron detector technology have played a pivotal role in enabling high-resolution structural studies by cryo-EM at 200 and 300 keV. Yet, theory and recent experiments indicate advantages to imaging at 100 keV, energies for which the current detectors have not been optimized. In this study, we evaluated the Gatan Alpine detector, designed for operation at 100 and 200 keV. Compared to the Gatan K3, Alpine demonstrated a significant DQE improvement at these energies, specifically a ∼ 4-fold improvement at Nyquist at 100 keV. In single-particle cryo-EM experiments, Alpine datasets yielded better than 2 Å resolution reconstructions of apoferritin at 120 and 200 keV on a ThermoFisher Scientific (TFS) Glacios microscope fitted with a non-standard SP-Twin lens. We also achieved a ∼ 3.2 Å resolution reconstruction of a 115 kDa asymmetric protein complex, proving Alpine's effectiveness with complex biological samples. In-depth analysis revealed that Alpine reconstructions are comparable to K3 reconstructions at 200 keV, and remarkably, reconstruction from Alpine at 120 keV on a TFS Glacios surpassed all but the 300 keV data from a TFS Titan Krios with GIF/K3. Additionally, we show Alpine's capability for high-resolution data acquisition and screening on lower-end systems by obtaining ∼ 3 Å resolution reconstructions of apoferritin and aldolase at 100 keV and detailed 2D averages of a 55 kDa sample using a side-entry cryo holder. Overall, we show that Gatan Alpine performs well with the standard 200 keV imaging systems and may potentially capture the benefits of lower accelerating voltages, bringing smaller sized particles within the scope of cryo-EM. | ||||||
History |
|
-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
---|
-Downloads & links
-Download
PDBx/mmCIF format | 8vsu.cif.gz | 334.1 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb8vsu.ent.gz | 266.1 KB | Display | PDB format |
PDBx/mmJSON format | 8vsu.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8vsu_validation.pdf.gz | 1.3 MB | Display | wwPDB validaton report |
---|---|---|---|---|
Full document | 8vsu_full_validation.pdf.gz | 1.3 MB | Display | |
Data in XML | 8vsu_validation.xml.gz | 38.9 KB | Display | |
Data in CIF | 8vsu_validation.cif.gz | 58.4 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/vs/8vsu ftp://data.pdbj.org/pub/pdb/validation_reports/vs/8vsu | HTTPS FTP |
-Related structure data
Related structure data | 43506MC M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
|
---|---|
1 |
|
-Components
#1: Protein | Mass: 41997.109 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: CAB39, MO25, CGI-66 / Cell line (production host): expi293T / Production host: Homo sapiens (human) / References: UniProt: Q9Y376 |
---|---|
#2: Protein | Mass: 50649.785 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: STK11, LKB1, PJS / Cell line (production host): expi293T / Production host: Homo sapiens (human) References: UniProt: Q15831, non-specific serine/threonine protein kinase |
#3: Protein | Mass: 47861.875 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: STRADA, LYK5, STRAD / Cell line (production host): expi293T / Production host: Homo sapiens (human) / References: UniProt: Q7RTN6 |
#4: Chemical | ChemComp-ADP / |
Has ligand of interest | Y |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT | |||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Molecular weight | Value: 0.1405 MDa / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: Homo sapiens (human) | |||||||||||||||||||||||||
Source (recombinant) | Organism: Homo sapiens (human) | |||||||||||||||||||||||||
Buffer solution | pH: 7.6 | |||||||||||||||||||||||||
Buffer component |
| |||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
---|---|
Microscopy | Model: TFS KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm |
Specimen holder | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron dose: 45.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1293 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
-Processing
EM software |
| ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CTF correction | Type: NONE | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 979927 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124780 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building |
| ||||||||||||||||||||||||
Atomic model building |
| ||||||||||||||||||||||||
Refine LS restraints |
|