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- PDB-9ot6: Cryo-EM structure of the PI4KA complex bound to an EFR3 interferi... -

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Basic information

Entry
Database: PDB / ID: 9ot6
TitleCryo-EM structure of the PI4KA complex bound to an EFR3 interfering nanobody (F3IN)
Components
  • EFR3 interfering Nanobody (F3IN)
  • Hyccin
  • Phosphatidylinositol 4-kinase alpha
  • Tetratricopeptide repeat protein 7B
KeywordsSIGNALING PROTEIN / PI4KA / TTC7B / FAM126A / Nanobody / Complex
Function / homology
Function and homology information


reorganization of cellular membranes to establish viral sites of replication / Synthesis of PIPs at the ER membrane / 1-phosphatidylinositol 4-kinase / 1-phosphatidylinositol 4-kinase activity / Synthesis of PIPs at the Golgi membrane / host-mediated perturbation of viral process / Golgi-associated vesicle membrane / phosphatidylinositol biosynthetic process / phosphatidylinositol-mediated signaling / phosphatidylinositol phosphate biosynthetic process ...reorganization of cellular membranes to establish viral sites of replication / Synthesis of PIPs at the ER membrane / 1-phosphatidylinositol 4-kinase / 1-phosphatidylinositol 4-kinase activity / Synthesis of PIPs at the Golgi membrane / host-mediated perturbation of viral process / Golgi-associated vesicle membrane / phosphatidylinositol biosynthetic process / phosphatidylinositol-mediated signaling / phosphatidylinositol phosphate biosynthetic process / myelination / protein localization to plasma membrane / neuron projection / cadherin binding / focal adhesion / signal transduction / extracellular exosome / ATP binding / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
PI4-kinase, N-terminal / PI4-kinase N-terminal region / Hyccin / Tetratricopeptide repeat protein 7, N-terminal / Hyccin / Tetratricopeptide repeat protein 7 N-terminal / : / Anaphase-promoting complex, cyclosome, subunit 3 / Tetratricopeptide repeat / Phosphoinositide 3-kinase family, accessory domain (PIK domain) ...PI4-kinase, N-terminal / PI4-kinase N-terminal region / Hyccin / Tetratricopeptide repeat protein 7, N-terminal / Hyccin / Tetratricopeptide repeat protein 7 N-terminal / : / Anaphase-promoting complex, cyclosome, subunit 3 / Tetratricopeptide repeat / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase family, accessory domain (PIK domain) / Phosphoinositide 3-kinase, accessory (PIK) domain superfamily / Phosphoinositide 3-kinase, accessory (PIK) domain / Phosphatidylinositol kinase / PIK helical domain profile. / Tetratricopeptide repeat / Phosphatidylinositol 3- and 4-kinases signature 1. / Phosphatidylinositol 3/4-kinase, conserved site / Phosphatidylinositol 3- and 4-kinases signature 2. / Phosphatidylinositol 3-/4-kinase, catalytic domain superfamily / Phosphoinositide 3-kinase, catalytic domain / Phosphatidylinositol 3- and 4-kinase / Phosphatidylinositol 3- and 4-kinases catalytic domain profile. / Phosphatidylinositol 3-/4-kinase, catalytic domain / TPR repeat region circular profile. / TPR repeat profile. / Tetratricopeptide repeats / Tetratricopeptide repeat / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / Protein kinase-like domain superfamily
Similarity search - Domain/homology
Phosphatidylinositol 4-kinase alpha / Tetratricopeptide repeat protein 7B / Hyccin
Similarity search - Component
Biological speciesHomo sapiens (human)
Lama glama (llama)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å
AuthorsShaw, A.L. / Suresh, S. / Yip, C.K. / Burke, J.E.
Funding support Canada, 4items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR)PJT-195808 Canada
Canadian Institutes of Health Research (CIHR)PJT-168907 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-03951 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)AWD-007855 Canada
CitationJournal: J Biol Chem / Year: 2025
Title: Development of an inhibitory TTC7B selective nanobody that blocks EFR3 recruitment of PI4KA.
Authors: Sushant Suresh / Alexandria L Shaw / Damilola K Akintola / Martine Lunke / Sophia Doerr / Pooja Rohilla / Tamas Balla / Calvin K Yip / Scott D Hansen / Jennifer A Cobb / John E Burke /
Abstract: Phosphatidylinositol 4 kinase IIIα (PI4KIIIα/PI4KA) is an essential lipid kinase that plays a critical role in regulating plasma membrane identity. PI4KA is primarily recruited to the plasma ...Phosphatidylinositol 4 kinase IIIα (PI4KIIIα/PI4KA) is an essential lipid kinase that plays a critical role in regulating plasma membrane identity. PI4KA is primarily recruited to the plasma membrane through the targeted recruitment by the proteins, EFR3A and EFR3B, which bind to the PI4KA accessory proteins TTC7 (TTC7A/B) and FAM126 (FAM126A/B). Here we characterised how both EFR3 isoforms interact with all possible TTC7-FAM126 combinations and developed a nanobody that specifically blocked EFR3-mediated PI4KA recruitment in TTC7B containing complexes. Most EFR3-TTC7-FAM126 combinations show similar binding affinities, with the exception of EFR3A-TTC7B-FAM126A, which binds with a ∼10-fold higher affinity. Moreover, we showed that EFR3B phosphorylation markedly decreased binding to TTC7-FAM126. Using a yeast display approach, we isolated a TTC7B selective nanobody that blocked EFR3 binding. Cryo-electron microscopy and hydrogen deuterium exchange mass spectrometry showed an extended interface with both PI4KA and TTC7B that sterically blocks EFR3 binding. The nanobody caused decreased membrane recruitment both on lipid bilayers and in cells, with decreased PM production of PI4P. Collectively, these findings provide new insights into PI4KA regulation and provide a tool for manipulating PI4KA complexes, that may be valuable for therapeutic targeting.
History
DepositionMay 26, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 3, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phosphatidylinositol 4-kinase alpha
B: Phosphatidylinositol 4-kinase alpha
C: EFR3 interfering Nanobody (F3IN)
D: Tetratricopeptide repeat protein 7B
E: Hyccin
F: Tetratricopeptide repeat protein 7B
G: Hyccin
H: EFR3 interfering Nanobody (F3IN)


Theoretical massNumber of molelcules
Total (without water)768,4098
Polymers768,4098
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phosphatidylinositol 4-kinase alpha / PI4-kinase alpha / PI4K-alpha / PtdIns-4-kinase alpha / Phosphatidylinositol 4-Kinase III alpha


Mass: 237246.438 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PI4KA, PIK4, PIK4CA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P42356, 1-phosphatidylinositol 4-kinase
#2: Antibody EFR3 interfering Nanobody (F3IN)


Mass: 18025.229 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Nanobody library in yeast surface display library / Source: (gene. exp.) Lama glama (llama) / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: Protein Tetratricopeptide repeat protein 7B / TPR repeat protein 7B / Tetratricopeptide repeat protein 7-like-1 / TPR repeat protein 7-like-1


Mass: 94294.109 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: TTC7B, TTC7L1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q86TV6
#4: Protein Hyccin / Down-regulated by CTNNB1 protein A


Mass: 34638.867 Da / Num. of mol.: 2 / Fragment: residues 1-308
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HYCC1, DRCTNNB1A, FAM126A / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9BYI3
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1Dimer of heterotetramers of PI4KA,TTC7B,FAM126A, and EFR3 interfering nanobody (F3IN)COMPLEXall0MULTIPLE SOURCES
2PI4KA,TTC7B,FAM126A complexCOMPLEX#1, #3-#41RECOMBINANT
3EFR3 interfering nanobody (F3IN)COMPLEX#21RECOMBINANT
Molecular weightValue: 0.767 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Spodoptera frugiperda (fall armyworm)7108
33Escherichia coli BL21(DE3) (bacteria)469008
Buffer solutionpH: 7
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMImidazoleC3H4N21
2150 mMSodium ChlorideNaCl1
35 %GlycerolC3H8O31
40.5 mMTris(2-carboxyethyl)phosphine1
SpecimenConc.: 0.75 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Gel filtration buffer, filtered through 0.22um filter and degassed. 0.75mg/ml of PI4KA with 3 fold molar excess F3IN Nanobody, with 0.25 mM BS3 crosslinker.
Specimen supportDetails: Glow discharged using the Pelco EasiGlow. 15mA Current.
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K / Details: Blot force -5, blot time 1.5 s.

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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: 165000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 14026
EM imaging opticsEnergyfilter name: TFS Selectris

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Processing

EM software
IDNameVersionCategory
4cryoSPARC4.5.2CTF correction
7UCSF ChimeraX1.8model fitting
9cryoSPARC4.5.2initial Euler assignment
10cryoSPARC4.5.2final Euler assignment
11cryoSPARC4.5.2classification
12cryoSPARC4.5.23D reconstruction
13PHENIX1.21_5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 282982 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model building

3D fitting-ID: 1

IDPDB-IDAccession codeDetailsInitial refinement model-IDSource nameType
19BAX

9bax

9BAXexcluding chain C and H1PDBexperimental model
2Nanobody F3INAlphaFoldin silico model
RefinementHighest resolution: 3.54 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00243590
ELECTRON MICROSCOPYf_angle_d0.47958990
ELECTRON MICROSCOPYf_dihedral_angle_d13.01416130
ELECTRON MICROSCOPYf_chiral_restr0.0366662
ELECTRON MICROSCOPYf_plane_restr0.0037438

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