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- EMDB-14077: Structure of PLA2R at pH 6.2 -

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Basic information

Entry
Database: EMDB / ID: EMD-14077
TitleStructure of PLA2R at pH 6.2
Map data
Sample
  • Organelle or cellular component: Secretory phospholipase A2 receptor
    • Protein or peptide: Secretory phospholipase A2 receptor
KeywordsMembranous nephropathy / IMMUNE SYSTEM
Function / homology
Function and homology information


negative regulation of arachidonic acid secretion / negative regulation of phospholipase A2 activity / Acyl chain remodelling of PG / Acyl chain remodelling of PC / Acyl chain remodelling of PI / positive regulation of DNA damage response, signal transduction by p53 class mediator / Acyl chain remodelling of PS / Acyl chain remodelling of PE / Synthesis of PA / positive regulation of podocyte apoptotic process ...negative regulation of arachidonic acid secretion / negative regulation of phospholipase A2 activity / Acyl chain remodelling of PG / Acyl chain remodelling of PC / Acyl chain remodelling of PI / positive regulation of DNA damage response, signal transduction by p53 class mediator / Acyl chain remodelling of PS / Acyl chain remodelling of PE / Synthesis of PA / positive regulation of podocyte apoptotic process / positive regulation of arachidonic acid secretion / oxidative stress-induced premature senescence / phospholipase binding / replicative senescence / receptor-mediated endocytosis / reactive oxygen species metabolic process / positive regulation of cytokine production / signaling receptor activity / carbohydrate binding / receptor complex / cell surface / extracellular region / plasma membrane
Similarity search - Function
Fibronectin type II domain / Fibronectin type II domain superfamily / Fibronectin type II domain / Fibronectin type-II collagen-binding domain signature. / Fibronectin type-II collagen-binding domain profile. / Fibronectin type 2 domain / C-type lectin, conserved site / C-type lectin domain signature. / Ricin-type beta-trefoil / Lectin domain of ricin B chain profile. ...Fibronectin type II domain / Fibronectin type II domain superfamily / Fibronectin type II domain / Fibronectin type-II collagen-binding domain signature. / Fibronectin type-II collagen-binding domain profile. / Fibronectin type 2 domain / C-type lectin, conserved site / C-type lectin domain signature. / Ricin-type beta-trefoil / Lectin domain of ricin B chain profile. / Ricin B, lectin domain / Ricin B-like lectins / Lectin C-type domain / C-type lectin domain profile. / C-type lectin-like / C-type lectin (CTL) or carbohydrate-recognition domain (CRD) / C-type lectin-like/link domain superfamily / C-type lectin fold
Similarity search - Domain/homology
: / Secretory phospholipase A2 receptor
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLockhart-Cairns MP / Rhoden SJ / Fresquet M / Jowitt TA / Briggs DC / Baldock C / Brenchley P / Lennon R
Funding support United Kingdom, 5 items
OrganizationGrant numberCountry
Kidney Research UKRP31/2015 United Kingdom
Kidney Research UKST8/2015 United Kingdom
European Union (EU)30560 United Kingdom
Wellcome Trust097820 United Kingdom
Wellcome Trust202860/Z/16/Z United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2022
Title: Structure of PLA2R reveals presentation of the dominant membranous nephropathy epitope and an immunogenic patch.
Authors: Maryline Fresquet / Michael P Lockhart-Cairns / Samuel J Rhoden / Thomas A Jowitt / David C Briggs / Clair Baldock / Paul E Brenchley / Rachel Lennon /
Abstract: Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most ...Membranous nephropathy is an autoimmune kidney disease caused by autoantibodies targeting antigens present on glomerular podocytes, instigating a cascade leading to glomerular injury. The most prevalent circulating autoantibodies in membranous nephropathy are against phospholipase A2 receptor (PLA2R), a cell surface receptor. The dominant epitope in PLA2R is located within the cysteine-rich domain, yet high-resolution structure-based mapping is lacking. In this study, we define the key nonredundant amino acids in the dominant epitope of PLA2R involved in autoantibody binding. We further describe two essential regions within the dominant epitope and spacer requirements for a synthetic peptide of the epitope for drug discovery. In addition, using cryo-electron microscopy, we have determined the high-resolution structure of PLA2R to 3.4 Å resolution, which shows that the dominant epitope and key residues within the cysteine-rich domain are accessible at the cell surface. In addition, the structure of PLA2R not only suggests a different orientation of domains but also implicates a unique immunogenic signature in PLA2R responsible for inducing autoantibody formation and recognition.
History
DepositionDec 23, 2021-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14077.png

Downloads & links

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Map

FileDownload / File: emd_14077.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.17 Å/pix.
x 256 pix.
= 298.803 Å		1.17 Å/pix.
x 256 pix.
= 298.803 Å		1.17 Å/pix.
x 256 pix.
= 298.803 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1672 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.227
Minimum - Maximum-0.42695343 - 1.3637415
Average (Standard dev.)-0.000081828715 (±0.036338206)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 298.8032 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_14077_additional_1.map
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Additional map: #2

Fileemd_14077_additional_2.map
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Half map: #1

Fileemd_14077_half_map_1.map
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Half map: #2

Fileemd_14077_half_map_2.map
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Sample components

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Entire : Secretory phospholipase A2 receptor

EntireName: Secretory phospholipase A2 receptor
Components
  • Organelle or cellular component: Secretory phospholipase A2 receptor
    • Protein or peptide: Secretory phospholipase A2 receptor

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Supramolecule #1: Secretory phospholipase A2 receptor

SupramoleculeName: Secretory phospholipase A2 receptor / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Truncated protein produced in HEK293 EBNA cells and purified in pH 6.2 buffer.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 180 KDa

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Macromolecule #1: Secretory phospholipase A2 receptor

MacromoleculeName: Secretory phospholipase A2 receptor / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EGVAAALTPE RLLEWQDKGI FVIQSESLKK CIQAGKSVLT LENCKQANKH MLWKWVSNHG LFNIGGSGC LGLNFSAPEQ PLSLYECDST LVSLRWRCNR KMITGPLQYS VQVAHDNTVV A SRKYIHKW ISYGSGGGDI CEYLHKDLHT IKGNTHGMPC MFPFQYNHQW ...String:
EGVAAALTPE RLLEWQDKGI FVIQSESLKK CIQAGKSVLT LENCKQANKH MLWKWVSNHG LFNIGGSGC LGLNFSAPEQ PLSLYECDST LVSLRWRCNR KMITGPLQYS VQVAHDNTVV A SRKYIHKW ISYGSGGGDI CEYLHKDLHT IKGNTHGMPC MFPFQYNHQW HHECTREGRE DD LLWCATT SRYERDEKWG FCPDPTSAEV GCDTIWEKDL NSHICYQFNL LSSLSWSEAH SSC QMQGGT LLSITDETEE NFIREHMSSK TVEVWMGLNQ LDEHAGWQWS DGTPLNYLNW SPEV NFEPF VEDHCGTFSS FMPSAWRSRD CESTLPYICK KYLNHIDHEI VEKDAWKYYA THCEP GWNP YNRNCYKLQK EEKTWHEALR SCQADNSALI DITSLAEVEF LVTLLGDENA SETWIG LSS NKIPVSFEWS NDSSVIFTNW HTLEPHIFPN RSQLCVSAEQ SEGHWKVKNC EERLFYI CK KAGHVLSDAE SGCQEGWERH GGFCYKIDTV LRSFDQASSG YYCPPALVTI TNRFEQAF I TSLISSVVKM KDSYFWIALQ DQNDTGEYTW KPVGQKPEPV QYTHWNTHQP RYSGGCVAM RGRHPLGRWE VKHCRHFKAM SLCKQPVENQ EKAEYEERWP FHPCYLDWES EPGLASCFKV FHSEKVLMK RTWREAEAFC EEFGAHLASF AHIEEENFVN ELLHSKFNWT EERQFWIGFN K RNPLNAGS WEWSDRTPVV SSFLDNTYFG EDARNCAVYK ANKTLLPLHC GSKREWICKI PR DVKPKIP FWYQYDVPWL FYQDAEYLFH TFASEWLNFE FVCSWLHSDL LTIHSAHEQE FIH SKIKAL SKYGASWWIG LQEERANDEF RWRDGTPVIY QNWDTGRERT VNNQSQRCGF ISSI TGLWG SEECSVSMPS ICKRKKVWLI EKKKDTPKQH GTCPKGWLYF NYKCLLLNIP KDPSS WKNW THAQHFCAEE GGTLVAIESE VEQAFITMNL FGQTTSVWIG LQNDDYETWL NGKPVV YSN WSPFDIINIP SHNTTEVQKH IPLCALLSSN PNFHFTGKWY FEDCGKEGYG FVCEKMQ DT SGHGVNTSDM YPMPNTLEYG NRTYKIINAN MTWYAAIKTC LMHKAQLVSI TDQYHQSF L TVVLNRLGYA HWIGLFTTDN GLNFDWSDGT KSSFTFWKDE ESSLLGDCVF ADSNGRWHS TACESFLQGA ICHVPPETRQ SEHPELCSET SIPWIKFKSN CYSFSTVLDS MSFEAAHEFC KKEGSNLLT IKDEAENAFL LEELFAFGSS VQMVWLNAQF DGNNETIKWF DGTPTDQSNW G IRKPDTDY FKPHHCVALR IPEGLWQLSP CQEKKGFICK MEADIHTAEA LPEKGPSHS

UniProtKB: UNIPROTKB: Q1301

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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

Concentration0.33 mg/mL
BufferpH: 6.2
Component:
ConcentrationFormulaName
10.0 mMC8H19NO5Bis-trisBis-tris methane
150.0 mMNaClSodium chlorideSodium Chloride

Details: 10 mM Bis-Tris, pH 6.2, 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Sample were applied to the grid in the chamber and blotted for 3 seconds..
DetailsSample was purified from secreted mammalian media with Ni affinity. The sample was further purified with SEC and buffer exchanged to pH 6.2.

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Details30 degree tilt of stage
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average exposure time: 2.0 sec. / Average electron dose: 42.9 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 950000
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 5 / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 250000

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7qsr:
CryoEM structure of the Ectodomain of Human PLA2R

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