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- PDB-8rrx: Structure of RyR1 reconstituted into lipid nanodisc in primed sta... -

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Basic information

Entry
Database: PDB / ID: 8rrx
TitleStructure of RyR1 reconstituted into lipid nanodisc in primed state in complex with Ca2+, ATP, caffeine and Nb9657
Components
  • Nanobody 9657
  • Peptidyl-prolyl cis-trans isomerase FKBP1B
  • Ryanodine receptor 1
KeywordsTRANSPORT PROTEIN / Ion channel / Ca2+ / tetramer
Function / homology
Function and homology information


ATP-gated ion channel activity / terminal cisterna / ryanodine receptor complex / ryanodine-sensitive calcium-release channel activity / release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / ossification involved in bone maturation / skin development / organelle membrane / cellular response to caffeine / outflow tract morphogenesis ...ATP-gated ion channel activity / terminal cisterna / ryanodine receptor complex / ryanodine-sensitive calcium-release channel activity / release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / ossification involved in bone maturation / skin development / organelle membrane / cellular response to caffeine / outflow tract morphogenesis / intracellularly gated calcium channel activity / regulation of ryanodine-sensitive calcium-release channel activity / toxic substance binding / voltage-gated calcium channel activity / smooth endoplasmic reticulum / regulation of cardiac muscle contraction by regulation of the release of sequestered calcium ion / skeletal muscle fiber development / striated muscle contraction / regulation of release of sequestered calcium ion into cytosol by sarcoplasmic reticulum / muscle contraction / release of sequestered calcium ion into cytosol / sarcoplasmic reticulum membrane / cellular response to calcium ion / sarcoplasmic reticulum / peptidylprolyl isomerase / peptidyl-prolyl cis-trans isomerase activity / calcium ion transmembrane transport / calcium channel activity / sarcolemma / Z disc / intracellular calcium ion homeostasis / disordered domain specific binding / protein homotetramerization / transmembrane transporter binding / calmodulin binding / intracellular membrane-bounded organelle / calcium ion binding / ATP binding / identical protein binding / membrane
Similarity search - Function
: / Ryanodine receptor junctional solenoid repeat / Ryanodine receptor, SPRY domain 2 / Ryanodine Receptor TM 4-6 / Ryanodine receptor / Ryanodine receptor, SPRY domain 1 / Ryanodine receptor, SPRY domain 3 / Ryanodine Receptor TM 4-6 / Ryanodine receptor Ryr / RyR domain ...: / Ryanodine receptor junctional solenoid repeat / Ryanodine receptor, SPRY domain 2 / Ryanodine Receptor TM 4-6 / Ryanodine receptor / Ryanodine receptor, SPRY domain 1 / Ryanodine receptor, SPRY domain 3 / Ryanodine Receptor TM 4-6 / Ryanodine receptor Ryr / RyR domain / : / RyR/IP3 receptor binding core, RIH domain superfamily / : / RyR/IP3R Homology associated domain / Inositol 1,4,5-trisphosphate/ryanodine receptor / RIH domain / RyR and IP3R Homology associated / Inositol 1,4,5-trisphosphate/ryanodine receptor / RIH domain / MIR motif / MIR domain / MIR domain profile. / Domain in ryanodine and inositol trisphosphate receptors and protein O-mannosyltransferases / Mir domain superfamily / SPRY domain / B30.2/SPRY domain / B30.2/SPRY domain profile. / SPRY domain / B30.2/SPRY domain superfamily / Domain in SPla and the RYanodine Receptor. / FKBP-type peptidyl-prolyl cis-trans isomerase domain profile. / FKBP-type peptidyl-prolyl cis-trans isomerase domain / FKBP-type peptidyl-prolyl cis-trans isomerase / Peptidyl-prolyl cis-trans isomerase domain superfamily / Ion transport domain / Ion transport protein / EF-hand domain pair / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / CAFFEINE / Chem-POV / Ryanodine receptor 1 / Peptidyl-prolyl cis-trans isomerase FKBP1B
Similarity search - Component
Biological speciesVicugna pacos (alpaca)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLi, C. / Efremov, R.G.
Funding support Belgium, European Union, 3items
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 23, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 9, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
H: Peptidyl-prolyl cis-trans isomerase FKBP1B
I: Ryanodine receptor 1
K: Nanobody 9657
L: Peptidyl-prolyl cis-trans isomerase FKBP1B
A: Ryanodine receptor 1
C: Nanobody 9657
J: Peptidyl-prolyl cis-trans isomerase FKBP1B
D: Ryanodine receptor 1
E: Nanobody 9657
F: Peptidyl-prolyl cis-trans isomerase FKBP1B
G: Ryanodine receptor 1
M: Nanobody 9657
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,373,16632
Polymers2,366,89812
Non-polymers6,26820
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 8 molecules HLJFIADG

#1: Protein
Peptidyl-prolyl cis-trans isomerase FKBP1B / PPIase FKBP1B / 12.6 kDa FK506-binding protein / 12.6 kDa FKBP / FKBP-12.6 / FK506-binding protein ...PPIase FKBP1B / 12.6 kDa FK506-binding protein / 12.6 kDa FKBP / FKBP-12.6 / FK506-binding protein 1B / FKBP-1B / Immunophilin FKBP12.6 / Rotamase


Mass: 11667.305 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: Q8HYX6, peptidylprolyl isomerase
#2: Protein
Ryanodine receptor 1 / RYR-1 / RyR1 / Skeletal muscle calcium release channel / Skeletal muscle ryanodine receptor / ...RYR-1 / RyR1 / Skeletal muscle calcium release channel / Skeletal muscle ryanodine receptor / Skeletal muscle-type ryanodine receptor / Type 1 ryanodine receptor


Mass: 564931.750 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P11716

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Antibody , 1 types, 4 molecules KCEM

#3: Antibody
Nanobody 9657


Mass: 15125.495 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Production host: Escherichia coli (E. coli)

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Non-polymers , 5 types, 20 molecules

#4: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#5: Chemical
ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC


Mass: 760.076 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#6: Chemical
ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#7: Chemical
ChemComp-CFF / CAFFEINE / 3,7-DIHYDRO-1,3,7-TRIMETHYL-1H-PURINE-2,6-DIONE


Mass: 194.191 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C8H10N4O2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#8: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Ryanodine receptor 1 complex with nanobody and FKBP12COMPLEX#1-#30MULTIPLE SOURCES
2Ryanodine receptor 1 complex with FKBP12COMPLEX#1-#21NATURAL
3Nanobody 9657COMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Oryctolagus cuniculus (rabbit)9986
33Vicugna pacos (alpaca)30538
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: JEOL CRYOSPECPORTER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.3.0particle selectiontemplate picking was used for particle picking
4RELION4.1CTF correction
10cryoSPARC3initial Euler assignment
12RELION3.1classification
13cryoSPARC4.313D reconstruction
Image processingDetails: The movies were selected after MotionCor correction and CTF refinement with the parameters: total drift less than 30 angstrom and resolution better than 5 angstrom
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2233299
Details: Particles were picked by template picking in Cryosparc
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 175535 / Algorithm: FOURIER SPACE / Symmetry type: POINT

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