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- EMDB-19117: Cryo-EM structure of the R243C mutant of human Prolyl Endopeptida... -

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Basic information

Entry
Database: EMDB / ID: EMD-19117
TitleCryo-EM structure of the R243C mutant of human Prolyl Endopeptidase-Like (PREPL) protein involved in Congenital myasthenic syndrome-22 (CMS22)
Map data
Sample
  • Complex: cryoEM structure of the R243C mutant of prolyl endopeptidase-like (PREPL) protein
    • Protein or peptide: Prolyl endopeptidase-like
Keywordsthio-esterase / HYDROLASE
Function / homology
Function and homology information


Golgi to plasma membrane protein transport / retrograde transport, endosome to Golgi / regulation of synaptic vesicle exocytosis / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / trans-Golgi network / peptidase activity / cytoskeleton / serine-type endopeptidase activity / Golgi apparatus / mitochondrion ...Golgi to plasma membrane protein transport / retrograde transport, endosome to Golgi / regulation of synaptic vesicle exocytosis / Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases / trans-Golgi network / peptidase activity / cytoskeleton / serine-type endopeptidase activity / Golgi apparatus / mitochondrion / proteolysis / nucleus / cytosol
Similarity search - Function
: / Peptidase S9A, prolyl oligopeptidase / Peptidase S9A, N-terminal domain / Prolyl oligopeptidase, N-terminal beta-propeller domain / Peptidase S9, prolyl oligopeptidase, catalytic domain / Prolyl oligopeptidase family / Alpha/Beta hydrolase fold
Similarity search - Domain/homology
Prolyl endopeptidase-like
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.01 Å
AuthorsTheodoropoulou A / Cavani E / Antanasijevic A / Marcaida MJ / Dal Peraro M
Funding support Switzerland, 1 items
OrganizationGrant numberCountry
Swiss National Science Foundation Switzerland
CitationJournal: JCI Insight / Year: 2024
Title: Missense variants in CMS22 patients reveal that PREPL has both enzymatic and nonenzymatic functions.
Authors: Yenthe Monnens / Anastasia Theodoropoulou / Karen Rosier / Kritika Bhalla / Alexia Mahy / Roeland Vanhoutte / Sandra Meulemans / Edoardo Cavani / Aleksandar Antanasijevic / Irma Lemmens / ...Authors: Yenthe Monnens / Anastasia Theodoropoulou / Karen Rosier / Kritika Bhalla / Alexia Mahy / Roeland Vanhoutte / Sandra Meulemans / Edoardo Cavani / Aleksandar Antanasijevic / Irma Lemmens / Jennifer A Lee / Catherine J Spellicy / Richard J Schroer / Ricardo A Maselli / Chamindra G Laverty / Patrizia Agostinis / David J Pagliarini / Steven Verhelst / Maria J Marcaida / Anne Rochtus / Matteo Dal Peraro / John Wm Creemers /
Abstract: Congenital myasthenic syndrome-22 (CMS22, OMIM 616224) is a rare genetic disorder caused by deleterious genetic variation in the prolyl endopeptidase-like (PREPL) gene. Previous reports have ...Congenital myasthenic syndrome-22 (CMS22, OMIM 616224) is a rare genetic disorder caused by deleterious genetic variation in the prolyl endopeptidase-like (PREPL) gene. Previous reports have described patients with deletions and nonsense variants in PREPL, but nothing is known about the effect of missense variants in the pathology of CMS22. In this study, we have functionally characterized missense variants in PREPL from 3 patients with CMS22, all with hallmark phenotypes. Biochemical evaluation revealed that these missense variants do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria and disease mechanism. Structural analysis showed that the variants affect regions most likely involved in intraprotein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the 3 variants. The importance of nonhydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines, which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the transgolgi network. In conclusion, these studies showed that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense variants that do not necessarily result in a loss of hydrolytic activity of PREPL.
History
DepositionDec 12, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19117.png

Downloads & links

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Map

FileDownload / File: emd_19117.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
0.93 Å/pix.
x 192 pix.
= 177.638 Å		0.93 Å/pix.
x 192 pix.
= 177.638 Å		0.93 Å/pix.
x 192 pix.
= 177.638 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9252 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.28076097 - 0.46403787
Average (Standard dev.)0.00016275034 (±0.012739886)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 177.6384 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19117_msk_1.map
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Half map: #2

Fileemd_19117_half_map_1.map
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Half map: #1

Fileemd_19117_half_map_2.map
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Sample components

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Entire : cryoEM structure of the R243C mutant of prolyl endopeptidase-like...

EntireName: cryoEM structure of the R243C mutant of prolyl endopeptidase-like (PREPL) protein
Components
  • Complex: cryoEM structure of the R243C mutant of prolyl endopeptidase-like (PREPL) protein
    • Protein or peptide: Prolyl endopeptidase-like

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Supramolecule #1: cryoEM structure of the R243C mutant of prolyl endopeptidase-like...

SupramoleculeName: cryoEM structure of the R243C mutant of prolyl endopeptidase-like (PREPL) protein
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Prolyl endopeptidase-like

MacromoleculeName: Prolyl endopeptidase-like / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Serine endopeptidases
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 73.477398 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SMDAFEKVRT KLETQPQEEY EIINVEVKHG GFVYYQEGCC LVRSKDEEAD NDNYEVLFNL EELKLDQPFI DCIRVAPDEK YVAAKIRTE DSEASTCVII KLSDQPVMEA SFPNVSSFEW VKDEEDEDVL FYTFQRNLRC HDVYRATFGD NKRNECFYTE K DPSYFVFL ...String:
SMDAFEKVRT KLETQPQEEY EIINVEVKHG GFVYYQEGCC LVRSKDEEAD NDNYEVLFNL EELKLDQPFI DCIRVAPDEK YVAAKIRTE DSEASTCVII KLSDQPVMEA SFPNVSSFEW VKDEEDEDVL FYTFQRNLRC HDVYRATFGD NKRNECFYTE K DPSYFVFL YLTKDSRFLT INIMNKTTSE VWLIDGLSPW DPPVLIQKRI HGVLYYVEHR DDELYILTNV GEPTEFKLMR TA ADTPAIM NWDLFFTMKR NTKVIDLDMF KDHCVLFLKH SNLLYVNVIG LADDSVRSLK LPPWACGFIM DTNSDPKNCP FQL CSPIRP PKYYTYKFAE GKLFEETGHE DPITKTSRVL RLEAKSKDGK LVPMTVFHKT DSEDLQKKPL LVHVYGAYGM DLKM NFRPE RRVLVDDGWI LAYCHVRGGG ELGLQWHADG RLTKKLNGLA DLEACIKTLH GQGFSQPSLT TLTAFSAGGV LAGAL CNSN PELVRAVTLE APFLDVLNTM MDTTLPLTLE ELEEWGNPSS DEKHKNYIKR YCPYQNIKPQ HYPSIHITAY ENDERV PLK GIVSYTEKLK EAIAEHAKDT GEGYQTPNII LDIQPGGNHV IEDSHKKITA QIKFLYEELG LDSTSVFEDL KKYLKF

UniProtKB: Prolyl endopeptidase-like

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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

Concentration13 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHepes2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid
1.0 mMTCEPTris-(2-Carboxyethyl)phosphine, Hydrochloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 4 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.01 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 137436
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
FSC plot (resolution estimation)

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