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- EMDB-70826: Cryo-EM structure of the PI4KA complex bound to an EFR3 interferi... -

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Basic information

Entry
Database: EMDB / ID: EMD-70826
TitleCryo-EM structure of the PI4KA complex bound to an EFR3 interfering nanobody (F3IN)
Map dataComposite map
Sample
  • Complex: Dimer of heterotetramers of PI4KA,TTC7B,FAM126A, and EFR3 interfering nanobody (F3IN)
    • Complex: PI4KA,TTC7B,FAM126A complex
      • Protein or peptide: Phosphatidylinositol 4-kinase alpha
      • Protein or peptide: Tetratricopeptide repeat protein 7B
      • Protein or peptide: Hyccin
    • Complex: EFR3 interfering nanobody (F3IN)
      • Protein or peptide: EFR3 interfering Nanobody (F3IN)
KeywordsPI4KA / TTC7B / FAM126A / Nanobody / Complex / SIGNALING PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.54 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_70826.png

Downloads & links

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Map

FileDownload / File: emd_70826.map.gz / Format: CCP4 / Size: 669.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComposite 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: 0.0371
Minimum - Maximum-0.034302045 - 0.21025667
Average (Standard dev.)0.0010623881 (±0.0056027896)
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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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
      • Protein or peptide: Phosphatidylinositol 4-kinase alpha
      • Protein or peptide: Tetratricopeptide repeat protein 7B
      • Protein or peptide: Hyccin
    • Complex: EFR3 interfering nanobody (F3IN)
      • Protein or peptide: 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 / Macromolecule list: all
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 / Macromolecule list: #1, #3-#4
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 / Macromolecule list: #2
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Phosphatidylinositol 4-kinase alpha

MacromoleculeName: Phosphatidylinositol 4-kinase alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: 1-phosphatidylinositol 4-kinase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 237.246438 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GSMAAAPARG GGGGGGGGGG CSGSGSSASR GFYFNTVLSL ARSLAVQRPA SLEKVQKLLC MCPVDFHGIF QLDERRRDAV IALGIFLIE SDLQHKDCVV PYLLRLLKGL PKVYWVEEST ARKGRGALPV AESFSFCLVT LLSDVAYRDP SLRDEILEVL L QVLHVLLG ...String:
GSMAAAPARG GGGGGGGGGG CSGSGSSASR GFYFNTVLSL ARSLAVQRPA SLEKVQKLLC MCPVDFHGIF QLDERRRDAV IALGIFLIE SDLQHKDCVV PYLLRLLKGL PKVYWVEEST ARKGRGALPV AESFSFCLVT LLSDVAYRDP SLRDEILEVL L QVLHVLLG MCQALEIQDK EYLCKYAIPC LIGISRAFGR YSNMEESLLS KLFPKIPPHS LRVLEELEGV RRRSFNDFRS IL PSNLLTV CQEGTLKRKT SSVSSISQVS PERGMPPPSS PGGSAFHYFE ASCLPDGTAL EPEYYFSTIS SSFSVSPLFN GVT YKEFNI PLEMLRELLN LVKKIVEEAV LKSLDAIVAS VMEANPSADL YYTSFSDPLY LTMFKMLRDT LYYMKDLPTS FVKE IHDFV LEQFNTSQGE LQKILHDADR IHNELSPLKL RCQANAACVD LMVWAVKDEQ GAENLCIKLS EKLQSKTSSK VIIAH LPLL ICCLQGLGRL CERFPVVVHS VTPSLRDFLV IPSPVLVKLY KYHSQYHTVA GNDIKISVTN EHSESTLNVM SGKKSQ PSM YEQLRDIAID NICRCLKAGL TVDPVIVEAF LASLSNRLYI SQESDKDAHL IPDHTIRALG HIAVALRDTP KVMEPIL QI LQQKFCQPPS PLDVLIIDQL GCLVITGNQY IYQEVWNLFQ QISVKASSVV YSATKDYKDH GYRHCSLAVI NALANIAA N IQDEHLVDEL LMNLLELFVQ LGLEGKRASE RASEKGPALK ASSSAGNLGV LIPVIAVLTR RLPPIKEAKP RLQKLFRDF WLYSVLMGFA VEGSGLWPEE WYEGVCEIAT KSPLLTFPSK EPLRSVLQYN SAMKNDTVTP AELSELRSTI INLLDPPPEV SALINKLDF AMSTYLLSVY RLEYMRVLRS TDPDRFQVMF CYFEDKAIQK DKSGMMQCVI AVADKVFDAF LNMMADKAKT K ENEEELER HAQFLLVNFN HIHKRIRRVA DKYLSGLVDK FPHLLWSGTV LKTMLDILQT LSLSLSADIH KDQPYYDIPD AP YRITVPD TYEARESIVK DFAARCGMIL QEAMKWAPTV TKSHLQEYLN KHQNWVSGLS QHTGLAMATE SILHFAGYNK QNT TLGATQ LSERPACVKK DYSNFMASLN LRNRYAGEVY GMIRFSGTTG QMSDLNKMMV QDLHSALDRS HPQHYTQAMF KLTA MLISS KDCDPQLLHH LCWGPLRMFN EHGMETALAC WEWLLAGKDG VEVPFMREMA GAWHMTVEQK FGLFSAEIKE ADPLA ASEA SQPKPCPPEV TPHYIWIDFL VQRFEIAKYC SSDQVEIFSS LLQRSMSLNI GGAKGSMNRH VAAIGPRFKL LTLGLS LLH ADVVPNATIR NVLREKIYST AFDYFSCPPK FPTQGEKRLR EDISIMIKFW TAMFSDKKYL TASQLVPPDN QDTRSNL DI TVGSRQQATQ GWINTYPLSS GMSTISKKSG MSKKTNRGSQ LHKYYMKRRT LLLSLLATEI ERLITWYNPL SAPELELD Q AGENSVANWR SKYISLSEKQ WKDNVNLAWS ISPYLAVQLP ARFKNTEAIG NEVTRLVRLD PGAVSDVPEA IKFLVTWHT IDADAPELSH VLCWAPTDPP TGLSYFSSMY PPHPLTAQYG VKVLRSFPPD AILFYIPQIV QALRYDKMGY VREYILWAAS KSQLLAHQF IWNMKTNIYL DEEGHQKDPD IGDLLDQLVE EITGSLSGPA KDFYQREFDF FNKITNVSAI IKPYPKGDER K KACLSALS EVKVQPGCYL PSNPEAIVLD IDYKSGTPMQ SAAKAPYLAK FKVKRCGVSE LEKEGLRCRS DSEDECSTQE AD GQKISWQ AAIFKVGDDC RQDMLALQII DLFKNIFQLV GLDLFVFPYR VVATAPGCGV IECIPDCTSR DQLGRQTDFG MYD YFTRQY GDESTLAFQQ ARYNFIRSMA AYSLLLFLLQ IKDRHNGNIM LDKKGHIIHI DFGFMFESSP GGNLGWEPDI KLTD EMVMI MGGKMEATPF KWFMEMCVRG YLAVRPYMDA VVSLVTLMLD TGLPCFRGQT IKLLKHRFSP NMTEREAANF IMKVI QSCF LSNRSRTYDM IQYYQNDIPY

UniProtKB: Phosphatidylinositol 4-kinase alpha

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Macromolecule #2: EFR3 interfering Nanobody (F3IN)

MacromoleculeName: EFR3 interfering Nanobody (F3IN) / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Lama glama (llama)
Molecular weightTheoretical: 18.025229 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MKYLLPTAAA GLLLLAAQPA MAMAQVQLQE SGGGLVQAGG SLRLSCAASG TISASDYMGW YRQAPGKERE LVASIDGGGI TNYADSVKG RFTISRDNAK NTVYLQMNSL KPEDTAVYYC AVDWILARYN FVIHYYWGQG TQVTVSSGSY PYDVPDYALE H HHHHH

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Macromolecule #3: Tetratricopeptide repeat protein 7B

MacromoleculeName: Tetratricopeptide repeat protein 7B / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 94.294109 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MATKKAGSRL ETEIERCRSE CQWERIPELV KQLSAKLIAN DDMAELLLGE SKLEQYLKEH PLRQGASPRG PKPQLTEVRK HLTAALDRG NLKSEFLQES NLIMAKLNYV EGDYKEALNI YARVGLDDLP LTAVPPYRLR VIAEAYATKG LCLEKLPISS S TSNLHVDR ...String:
MATKKAGSRL ETEIERCRSE CQWERIPELV KQLSAKLIAN DDMAELLLGE SKLEQYLKEH PLRQGASPRG PKPQLTEVRK HLTAALDRG NLKSEFLQES NLIMAKLNYV EGDYKEALNI YARVGLDDLP LTAVPPYRLR VIAEAYATKG LCLEKLPISS S TSNLHVDR EQDVITCYEK AGDIALLYLQ EIERVILSNI QNRSPKPGPA PHDQELGFFL ETGLQRAHVL YFKNGNLTRG VG RFRELLR AVETRTTQNL RMTIARQLAE ILLRGMCEQS YWNPLEDPPC QSPLDDPLRK GANTKTYTLT RRARVYSGEN IFC PQENTE EALLLLLISE SMANRDAVLS RIPEHKSDRL ISLQSASVVY DLLTIALGRR GQYEMLSECL ERAMKFAFEE FHLW YQFAL SLMAAGKSAR AVKVLKECIR LKPDDATIPL LAAKLCMGSL HWLEEAEKFA KTVVDVGEKT SEFKAKGYLA LGLTY SLQA TDASLRGMQE VLQRKALLAF QRAHSLSPTD HQAAFYLALQ LAISRQIPEA LGYVRQALQL QGDDANSLHL LALLLS AQK HYHDALNIID MALSEYPENF ILLFSKVKLQ SLCRGPDEAL LTCKHMLQIW KSCYNLTNPS DSGRGSSLLD RTIADRR QL NTITLPDFSD PETGSVHATS VAASRVEQAL SEVASSLQSS APKQGPLHPW MTLAQIWLHA AEVYIGIGKP AEATACTQ E AANLFPMSHN VLYMRGQIAE LRGSMDEARR WYEEALAISP THVKSMQRLA LILHQLGRYS LAEKILRDAV QVNSTAHEV WNGLGEVLQA QGNDAAATEC FLTALELEAS SPAVPFTIIP RVL

UniProtKB: Tetratricopeptide repeat protein 7B

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Macromolecule #4: Hyccin

MacromoleculeName: Hyccin / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 34.638867 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MFTSEKGVVE EWLSEFKTLP ETSLPNYATN LKDKSSLVSS LYKVIQEPQS ELLEPVCHQL FEFYRSGEEQ LLQFTLQFLP ELIWCYLAV SASRNVHSSG CIEALLLGVY NLEIVDKQGH TKVLSFTIPS LSKPSVYHEP SSIGSMALTE SALSQHGLSK V VYSGPHPQ ...String:
MFTSEKGVVE EWLSEFKTLP ETSLPNYATN LKDKSSLVSS LYKVIQEPQS ELLEPVCHQL FEFYRSGEEQ LLQFTLQFLP ELIWCYLAV SASRNVHSSG CIEALLLGVY NLEIVDKQGH TKVLSFTIPS LSKPSVYHEP SSIGSMALTE SALSQHGLSK V VYSGPHPQ REMLTAQNRF EVLTFLLLCY NAALTYMPSV SLQSLCQICS RICVCGYPRQ HVRKYKGISS RIPVSSGFMV QM LTGIYFA FYNGEWDLAQ KALDDIIYRA QLELYPEPLL VANAIKASLP HGPMKSNKEG TRCIQVEITP T

UniProtKB: Hyccin

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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
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: Glow discharged using the Pelco EasiGlow. 15mA Current.
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 reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.54 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)

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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
Output model

PDB-9ot6:
Cryo-EM structure of the PI4KA complex bound to an EFR3 interfering nanobody (F3IN)

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