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- EMDB-19487: Structure of RyR1 reconstituted into lipid nanodisc in primed sta... -

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Basic information

Entry
Database: EMDB / ID: EMD-19487
TitleStructure of RyR1 reconstituted into lipid nanodisc in primed state in complex with Ca2+, ATP, caffeine and Nb9657
Map data
Sample
  • Complex: ryanodine receptor 1 in complex with FKBP12 and nanobody
    • Protein or peptide: Ryanodine receptor 1
KeywordsIon channel / Ca2+ / tetramer / TRANSPORT PROTEIN
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLi C / Efremov RG
Funding support Belgium, European Union, 3 items
OrganizationGrant numberCountry
Research Foundation - Flanders (FWO)G0H5916N Belgium
Research Foundation - Flanders (FWO)G054617N Belgium
European Research Council (ERC)726436European Union
CitationJournal: J Biol Chem / Year: 2024
Title: Rapid small-scale nanobody-assisted purification of ryanodine receptors for cryo-EM.
Authors: Chenyao Li / Katrien Willegems / Tomasz Uchański / Els Pardon / Jan Steyaert / Rouslan G Efremov /
Abstract: Ryanodine receptors (RyRs) are large Ca release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of ...Ryanodine receptors (RyRs) are large Ca release channels residing in the endoplasmic or sarcoplasmic reticulum membrane. Three isoforms of RyRs have been identified in mammals, the disfunction of which has been associated with a series of life-threatening diseases. The need for large amounts of native tissue or eukaryotic cell cultures limits advances in structural studies of RyRs. Here, we report a method that utilizes nanobodies to purify RyRs from only 5 mg of total protein. The purification process, from isolated membranes to cryo-EM grade protein, is achieved within 4 h on the bench, yielding protein usable for cryo-EM analysis. This is demonstrated by solving the structures of rabbit RyR1, solubilized in detergent, reconstituted into lipid nanodiscs or liposomes, and bovine RyR2 reconstituted in nanodisc, and mouse RyR2 in detergent. The reported method facilitates structural studies of RyRs directed toward drug development and is useful in cases where the amount of starting material is limited.
History
DepositionJan 25, 2024-
Header (metadata) releaseDec 4, 2024-
Map releaseDec 4, 2024-
UpdateDec 11, 2024-
Current statusDec 11, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19487.png

Downloads & links

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Map

FileDownload / File: emd_19487.map.gz / Format: CCP4 / Size: 347.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 450 pix.
= 511.65 Å		1.14 Å/pix.
x 450 pix.
= 511.65 Å		1.14 Å/pix.
x 450 pix.
= 511.65 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.137 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.1957326 - 2.1922054
Average (Standard dev.)0.0030620873 (±0.052623603)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions450450450
Spacing450450450
CellA=B=C: 511.65 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19487_msk_1.map
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Half map: #1

Fileemd_19487_half_map_1.map
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Half map: #2

Fileemd_19487_half_map_2.map
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Sample components

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Entire : ryanodine receptor 1 in complex with FKBP12 and nanobody

EntireName: ryanodine receptor 1 in complex with FKBP12 and nanobody
Components
  • Complex: ryanodine receptor 1 in complex with FKBP12 and nanobody
    • Protein or peptide: Ryanodine receptor 1

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Supramolecule #1: ryanodine receptor 1 in complex with FKBP12 and nanobody

SupramoleculeName: ryanodine receptor 1 in complex with FKBP12 and nanobody
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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Macromolecule #1: Ryanodine receptor 1

MacromoleculeName: Ryanodine receptor 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
SequenceString: VQFLRTDDEV VLQCSATVLK EQLKLCLAAE GFGNRLCFLE PTSNAQNVPP DLAICCFTLE QSLSVRALQE MLANTVEAGV ESSQGGGHR TLLYGHAILL RHAHSRMYLS CLTTSRSMTD KLAFDVGLQE DATGEACWWT MHPASKQRSE GEKVRVGDDL I LVSVSSER ...String:
VQFLRTDDEV VLQCSATVLK EQLKLCLAAE GFGNRLCFLE PTSNAQNVPP DLAICCFTLE QSLSVRALQE MLANTVEAGV ESSQGGGHR TLLYGHAILL RHAHSRMYLS CLTTSRSMTD KLAFDVGLQE DATGEACWWT MHPASKQRSE GEKVRVGDDL I LVSVSSER YLHLSTASGE LQVDASFMQT LWNMNPICSC CEEGYVTGGH VLRLFHGHMD ECLTISAADS DDQRRLVYYE GG AVCTHAR SLWRLEPLRI SWSGSHLRWG QPLRIRHVTT GRYLALTEDQ GLVVVDACKA HTKATSFCFR VSKEKLDTAP KRD VEGMGP PEIKYGESLC FVQHVASGLW LTYAAPDPKA LRLGVLKKKA ILHQEGHMDD ALFLTRCQQE ESQAARMIHS TAGL YNQFI KGLDSFSGKP RGSGPPAGPA LPIEAVILSL QDLIGYFEPP SEELQHEEKQ SKLRSLRNRQ SLFQEEGMLS LVLNC IDRL NVYTTAAHFA EYAGEEAAES WKEIVNLLYE LLASLIRGNR ANCALFSTNL DWVVSKLDRL EASSGILEVL YCVLIE SPE VLNIIQENHI KSIISLLDKH GRNHKVLDVL CSLCVCNGVA VRSNQDLITE NLLPGRELLL QTNLINYVTS IRPNIFV GR AEGSTQYGKW YFEVMVDEVV PFLTAQATHL RVGWALTEGY SPYPGGGEGW GGNGVGDDLY SYGFDGLHLW TGHVARPV T SPGQHLLAPE DVVSCCLDLS VPSISFRING CPVQGVFEAF NLDGLFFPVV SFSAGVKVRF LLGGRHGEFK FLPPPGYAP CHEAVLPRER LRLEPIKEYR REGPRGPHLV GPSRCLSHTD FVPCPVDTVQ IVLPPHLERI REKLAENIHE LWALTRIEQG WTYGPVRDD NKRLHPCLVN FHSLPEPERN YNLQMSGETL KTLLALGCHV GMADEKAEDN LKKTKLPKTY MMSNGYKPAP L DLSHVRLT PAQTTLVDRL AENGHNVWAR DRVAQGWSYS AVQDIPARRN PRLVPYRLLD EATKRSNRDS LCQAVRTLLG YG YNIEPPD QEPSQVENQS RWDRVRIFRA EKSYTVQSGR WYFEFEAVTT GEMRVGWARP ELRPDVELGA DELAYVFNGH RGQ RWHLGS EPFGRPWQSG DVVGCMIDLT ENTIIFTLNG EVLMSDSGSE TAFREIEIGD GFLPVCSLGP GQVGHLNLGQ DVSS LRFFA ICGLQEGFEP FAINMQRPVT TWFSKSLPQF EPVPPEHPHY EVARMDGTVD TPPCLRLAHR TWGSQNSLVE MLFLR LSLP VQFHQHFRCT AGATPLAPPG LQPPAEDEAR AAEPDPDYEN LRRSAGGWGE AEGGKEGTAK EGTPGGTPQP GVEAQP VRA ENEKDATTEK NKKRGFLFKA KKAAMMTQPP ATPALPRLPH DVVPADNRDD PEIILNTTTY YYSVRVFAGQ EPSCVWV GW VTPDYHQHDM NFDLSKVRAV TVTMGDEQGN VHSSLKCSNC YMVWGGDFVS PGQQGRISHT DLVIGCLVDL ATGLMTFT A NGKESNTFFQ VEPNTKLFPA VFVLPTHQNV IQFELGKQKN IMPLSAAMFL SERKNPAPQC PPRLEVQMLM PVSWSRMPN HFLQVETRRA GERLGWAVQC QDPLTMMALH IPEENRCMDI LELSERLDLQ RFHSHTLRLY RAVCALGNNR VAHALCSHVD QAQLLHALE DAHLPGPLRA GYYDLLISIH LESACRSRRS MLSEYIVPLT PETRAITLFP PGRKGGNARR HGLPGVGVTT S LRPPHHFS PPCFVAALPA AGVAEAPARL SPAIPLEALR DKALRMLGEA VRDGGQHARD PVGGSVEFQF VPVLKLVSTL LV MGIFGDE DVKQILKMIE PEVFTEEEEE EEEEEEEEEE EEEDEEEKEE DEEEEEKEDA EKEEEEAPEG EKEDLEEGLL QMK LPESVK LQMCNLLEYF CDQELQHRVE SLAAFAERYV DKLQANQRSR YALLMRAFTM SAAETARRTR EFRSPPQEQI NMLL HFKDE ADEEDCPLPE DIRQDLQDFH QDLLAHCGIQ LEGEEEEPEE ETSLSSRLRS LLETVRLVKK KEEKPEEELP AEEKK PQSL QELVSHMVVR WAQEDYVQSP ELVRAMFSLL HRQYDGLGEL LRALPRAYTI SPSSVEDTMS LLECLGQIRS LLIVQM GPQ EENLMIQSIG NIMNNKVFYQ HPNLMRALGM HETVMEVMVN VLGGGETKEI RFPKMVTSCC RFLCYFCRIS RQNQRSM FD HLSYLLENSG IGLGMQGSTP LDVAAASVID NNELALALQE QDLEKVVSYL AGCGLQSCPM LLAKGYPDIG WNPCGGER Y LDFLRFAVFV NGESVEENAN VVVRLLIRKP ECFGPALRGE GGSGLLAAIE EAIRISEDPA RDGPGVRRDR RREHFGEEP PEENRVHLGH AIMSFYAALI DLLGRCAPEM HLIQAGKGEA LRIRAILRSL VPLDDLVGII SLPLQIPTLG KDGALVQPKM SASFVPDHK ASMVLFLDRV YGIENQDFLL HVLDVGFLPD MRAAASLDTA TFSTTEMALA LNRYLCLAVL PLITKCAPLF A GTEHRAIM VDSMLHTVYR LSRGRSLTKA QRDVIEDCLM ALCRYIRPSM LQHLLRRLVF DVPILNEFAK MPLKLLTNHY ER CWKYYCL PTGWANFGVT SEEELHLTRK LFWGIFDSLA HKKYDQELYR MAMPCLCAIA GALPPDYVDA SYSSKAEKKA TVD AEGNFD PRPVETLNVI IPEKLDSFIN KFAEYTHEKW AFDKIQNNWS YGENVDEELK THPMLRPYKT FSEKDKEIYR WPIK ESLKA MIAWEWTIEK AREGEEERTE KKKTRKISQT AQTYDPREGY NPQPPDLSGV TLSRELQAMA EQLAENYHNT WGRKK KQEL EAKGGGTHPL LVPYDTLTAK EKARDREKAQ ELLKFLQMNG YAVTRGLKDM ELDTSSIEKR FAFGFLQQLL RWMDIS QEF IAHLEAVVSS GRVEKSPHEQ EIKFFAKILL PLINQYFTNH CLYFLSTPAK VLGSGGHASN KEKEMITSLF CKLAALV RH RVSLFGTDAP AVVNCLHILA RSLDARTVMK SGPEIVKAGL RSFFESASED IEKMVENLRL GKVSQARTQV KGVGQNLT Y TTVALLPVLT TLFQHIAQHQ FGDDVILDDV QVSCYRTLCS IYSLGTTKNT YVEKLRPALG ECLARLAAAM PVAFLEPQL NEYNACSVYT TKSPRERAIL GLPNSVEEMC PDIPVLDRLM ADIGGLAESG ARYTEMPHVI EITLPMLCSY LPRWWERGPE APPPALPAG APPPCTAVTS DHLNSLLGNI LRIIVNNLGI DEATWMKRLA VFAQPIVSRA RPELLHSHFI PTIGRLRKRA G KVVAEEEQ LRLEAKAEAE EGELLVRDEF SVLCRDLYAL YPLLIRYVDN NRAHWLTEPN ANAEELFRMV GEIFIYWSKS HN FKREEQN FVVQNEINNM SFLTADSKSK MAKAGDAQSG GSDQERTKKK RRGDRYSVQT SLIVATLKKM LPIGLNMCAP TDQ DLIMLA KTRYALKDTD EEVREFLQNN LHLQGKVEGS PSLRWQMALY RGLPGREEDA DDPEKIVRRV QEVSAVLYHL EQTE HPYKS KKAVWHKLLS KQRRRAVVAC FRMTPLYNLP THRACNMFLE SYKAAWILTE DHSFEDRMID DLSKAGEQEE EEEEV EEKK PDPLHQLVLH FSRTALTEKS KLDEDYLYMA YADIMAKSCH LEEGGENGEA EEEEVEVSFE EKEMEKQRLL YQQSRL HTR GAAEMVLQMI SACKGETGAM VSSTLKLGIS ILNGGNAEVQ QKMLDYLKDK KEVGFFQSIQ ALMQTCSVLD LNAFERQ NK AEGLGMVNED GTVINRQNGE KVMADDEFTQ DLFRFLQLLC EGHNNDFQNY LRTQTGNTTT INIIICTVDY LLRLQESI S DFYWYYSGKD VIEEQGKRNF SKAMSVAKQV FNSLTEYIQG PCTGNQQSLA HSRLWDAVVG FLHVFAHMMM KLAQDSSQI ELLKELLDLQ KDMVVMLLSL LEGNVVNGMI ARQMVDMLVE SSSNVEMILK FFDMFLKLKD IVGSEAFQDY VTDPRGLISK KDFQKAMDS QKQFTGPEIQ FLLSCSEADE NEMINFEEFA NRFQEPARDI GFNVAVLLTN LSEHVPHDPR LRNFLELAES I LEYFRPYL GRIEIMGASR RIERIYFEIS ETNRAQWEMP QVKESKRQFI FDVVNEGGEA EKMELFVSFC EDTIFEMQIA AQ ISEPEGE PEADEDEGMG EAAAEGAEEG AAGAEGAAGT VAAGATARLA AAAARALRGL SYRSLRRRVR RLRRLTAREA ATA LAALLW AVVARAGAAG AGAAAGALRL LWGSLFGGGL VEGAKKVTVT ELLAGMPDPT SDEVHGEQPA GPGGDADGAG EGEG EGDAA EGDGDEEVAG HEAGPGGAEG VVAVADGGPF RPEGAGGLGD MGDTTPAEPP TPEGSPILKR KLGVDGEEEE LVPEP EPEP EPEPEKADEE NGEKEEVPEA PPEPPKKAPP SPPAKKEEAG GAGMEFWGEL EVQRVKFLNY LSRNFYTLRF LALFLA FAI NFILLFYKVS DSPPGEDDME GSAAGDLAGA GSGGGSGWGS GAGEEAEGDE DENMVYYFLE ESTGYMEPAL WCLSLLH TL VAFLCIIGYN CLKVPLVIFK REKELARKLE FDGLYITEQP GDDDVKGQWD RLVLNTPSFP SNYWDKFVKR KVLDKHGD I FGRERIAELL GMDLASLEIT AHNERKPDPP PGLLTWLMSI DVKYQIWKFG VIFTDNSFLY LGWYMVMSLL GHYNNFFFA AHLLDIAMGV KTLRTILSSV THNGKQLVMT VGLLAVVVYL YTVVAFNFFR KFYNKSEDED EPDMKCDDMM TCYLFHMYVG VRAGGGIGD EIEDPAGDEY ELYRVVFDIT FFFFVIVILL AIIQGLIIDA FGELRDQQEQ VKEDMETKCF ICGIGSDYFD T TPHGFETH TLEEHNLANY MFFLMYLINK DETEHTGQES YVWKMYQERC WDFFPAGDCF RKQYEDQLS

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statetissue

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm

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Image processing

DetailsThe movies were selected after MotionCor correction and CTF refinement with the parameters: total drift less than 30 angstrom and resolution better than 5 angstrom
Particle selectionNumber selected: 2233299
Details: Particles were picked by template picking in Cryosparc
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7tzc

Final reconstructionNumber classes used: 3 / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.31) / Number images used: 175535
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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