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- EMDB-70825: Local Refinement B of PI4KA/TTC7B/FAM126A/F3IN nanbody -

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Basic information

Entry
Database: EMDB / ID: EMD-70825
TitleLocal Refinement B of PI4KA/TTC7B/FAM126A/F3IN nanbody
Map dataLocal Refinement B - Map
Sample
  • Complex: Dimer of heterotetramers of PI4KA,TTC7B,FAM126A, and EFR3 interfering nanobody (F3IN)
    • Complex: PI4KA,TTC7B,FAM126A complex
    • Complex: EFR3 interfering nanobody (F3IN)
KeywordsPI4KA / TTC7B / FAM126A / Nanobody / Complex / SIGNALING PROTEIN
Biological speciesHomo sapiens (human) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.77 Å
AuthorsShaw AL / Suresh S / Yip CK / Burke JE
Funding support Canada, 4 items
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, 2025-
Header (metadata) releaseDec 3, 2025-
Map releaseDec 3, 2025-
UpdateDec 3, 2025-
Current statusDec 3, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70825.png

Downloads & links

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Map

FileDownload / File: emd_70825.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal Refinement B - Map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.77 Å/pix.
x 560 pix.
= 431.2 Å		0.77 Å/pix.
x 560 pix.
= 431.2 Å		0.77 Å/pix.
x 560 pix.
= 431.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.77 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0323
Minimum - Maximum-0.12947108 - 0.1962074
Average (Standard dev.)-0.000003885397 (±0.0037932766)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions560560560
Spacing560560560
CellA=B=C: 431.19998 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Local Refinement B - Half Map B

Fileemd_70825_half_map_1.map
AnnotationLocal Refinement B - Half Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Local Refinement B - Half Map A

Fileemd_70825_half_map_2.map
AnnotationLocal Refinement B - Half Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimer of heterotetramers of PI4KA,TTC7B,FAM126A, and EFR3 interfe...

EntireName: Dimer of heterotetramers of PI4KA,TTC7B,FAM126A, and EFR3 interfering nanobody (F3IN)
Components
  • Complex: Dimer of heterotetramers of PI4KA,TTC7B,FAM126A, and EFR3 interfering nanobody (F3IN)
    • Complex: PI4KA,TTC7B,FAM126A complex
    • Complex: EFR3 interfering nanobody (F3IN)

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Supramolecule #1: Dimer of heterotetramers of PI4KA,TTC7B,FAM126A, and EFR3 interfe...

SupramoleculeName: Dimer of heterotetramers of PI4KA,TTC7B,FAM126A, and EFR3 interfering nanobody (F3IN)
type: complex / ID: 1 / Parent: 0
Molecular weightTheoretical: 767 KDa

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Supramolecule #2: PI4KA,TTC7B,FAM126A complex

SupramoleculeName: PI4KA,TTC7B,FAM126A complex / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: EFR3 interfering nanobody (F3IN)

SupramoleculeName: EFR3 interfering nanobody (F3IN) / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Lama glama (llama)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.75 mg/mL
BufferpH: 7
Component:
ConcentrationFormulaName
20.0 mMC3H4N2Imidazole
150.0 mMNaClSodium Chloride
5.0 %C3H8O3Glycerol
0.5 mMTris(2-carboxyethyl)phosphine
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force -5, blot time 1.5 s..
DetailsGel 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.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Number grids imaged: 2 / Number real images: 14026 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.5.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Details: PI4KA complex map without F3IN lowpass filtered to 20 Angstroms
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.5.2) / Number images used: 282982
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

9bax

source_name: PDB, initial_model_type: experimental modelexcluding chain C and H
source_name: AlphaFold, initial_model_type: in silico modelNanobody F3IN

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