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- PDB-8vsu: Cryo-EM structure of LKB1-STRADalpha-MO25alpha heterocomplex -

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Basic information

Entry
Database: PDB / ID: 8vsu
TitleCryo-EM structure of LKB1-STRADalpha-MO25alpha heterocomplex
Components
  • Calcium-binding protein 39
  • Isoform 3 of STE20-related kinase adapter protein alpha
  • Serine/threonine-protein kinase STK11
KeywordsTRANSFERASE / serine/threonine kinase / pseudokinase / complex
Function / homology
Function and homology information


positive regulation of vesicle transport along microtubule / intracellular protein-containing complex / Golgi localization / LRR domain binding / 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 / Golgi localization / LRR domain binding / 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 / epithelial cell proliferation involved in prostate gland development / response to thyroid hormone / Energy dependent regulation of mTOR by LKB1-AMPK / positive thymic T cell selection / vasculature development / regulation of Wnt signaling pathway / anoikis / response to glucagon / negative regulation of cold-induced thermogenesis / FOXO-mediated transcription of cell death genes / positive regulation of axonogenesis / regulation of dendrite morphogenesis / establishment of cell polarity / G1 to G0 transition / response to ionizing radiation / cellular response to UV-B / response to lipid / activation of protein kinase activity / positive regulation of transforming growth factor beta receptor signaling pathway / protein kinase activator activity / regulation of phosphatidylinositol 3-kinase/protein kinase B signal transduction / 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 serine/threonine kinase activator activity / protein dephosphorylation / 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 / autophagy / kinase binding / Z disc / 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 / non-specific serine/threonine protein kinase / regulation of cell cycle / intracellular signal transduction / negative regulation of cell population proliferation / protein phosphorylation / 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 / cytoplasm / cytosol
Similarity search - Function
Serine/Threonine kinase LKB1, catalytic domain / : / Mo25-like / Mo25-like / Armadillo-like helical / Armadillo-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain ...Serine/Threonine kinase LKB1, catalytic domain / : / Mo25-like / Mo25-like / Armadillo-like helical / Armadillo-type fold / Serine/threonine-protein kinase, active site / Serine/Threonine protein kinases active-site signature. / Protein kinase domain / Serine/Threonine protein kinases, catalytic domain / Protein kinase, ATP binding site / Protein kinases ATP-binding region signature. / Protein kinase domain profile. / Protein kinase domain / Protein kinase-like domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Serine/threonine-protein kinase STK11 / STE20-related kinase adapter protein alpha / Calcium-binding protein 39
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.86 Å
AuthorsChan, L.M. / Courteau, B.J. / Verba, K.A.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: 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 / Hevatib 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 / Hevatib 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 voltages, 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 for a 115 kDa asymmetric protein complex, proving its 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, possibly bringing smaller sized particles within the scope of cryo-EM.
History
DepositionJan 24, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Calcium-binding protein 39
C: Serine/threonine-protein kinase STK11
B: Isoform 3 of STE20-related kinase adapter protein alpha
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,9364
Polymers140,5093
Non-polymers4271
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Calcium-binding protein 39 / MO25alpha / Protein Mo25


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 Serine/threonine-protein kinase STK11 / Liver kinase B1 / LKB1 / hLKB1 / Renal carcinoma antigen NY-REN-19


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 Isoform 3 of STE20-related kinase adapter protein alpha / STRAD alpha / STE20-related adapter protein / Serologically defined breast cancer antigen NY-BR-96


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 / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Heterotrimeric complex of serine/threonine kinase LKB1 with psuedokinase STRADa and scaffolding MO25a
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.1405 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris HCl1
2350 mMsodium chlorideNaCl1
35 %glycerol1
40.5 mMDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron 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 opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameCategoryFitting-ID
7UCSF ChimeraXmodel fitting1
14Rosettamodel fitting2
15ISOLDEmodel refinement2
CTF correctionType: NONE
Particle selectionNum. of particles selected: 979927
3D reconstructionResolution: 2.86 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124780 / Symmetry type: POINT
Atomic model building
IDProtocol
1RIGID BODY FIT
2FLEXIBLE FIT
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
12WTK

2wtk

12WTK1PDBexperimental model
22
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0127678
ELECTRON MICROSCOPYf_angle_d1.48910374
ELECTRON MICROSCOPYf_dihedral_angle_d11.5852917
ELECTRON MICROSCOPYf_chiral_restr0.0731148
ELECTRON MICROSCOPYf_plane_restr0.0111326

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