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- PDB-7tdi: Rabbit RyR1 with AMP-PCP and high Ca2+ embedded in nanodisc in cl... -

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Basic information

Entry
Database: PDB / ID: 7tdi
TitleRabbit RyR1 with AMP-PCP and high Ca2+ embedded in nanodisc in closed-inactivated conformation class 2 (Dataset-A)
ComponentsRyanodine receptor 1,Ryanodine receptor 1,RyR1
KeywordsTRANSPORT PROTEIN / Ryanodine Receptor / RyR1 / Intracellular Calcium channel / Ca2+ / Inactivation / Excitation-Contraction coupling
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 / toxic substance binding / voltage-gated calcium channel activity / smooth endoplasmic reticulum / skeletal muscle fiber development / striated muscle contraction / release of sequestered calcium ion into cytosol / muscle contraction / sarcoplasmic reticulum membrane / cellular response to calcium ion / sarcoplasmic reticulum / 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 / calcium ion binding / ATP binding / identical protein binding / membrane
Similarity search - Function
Ryanodine receptor, SPRY domain 2 / : / Ryanodine receptor junctional solenoid repeat / 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, SPRY domain 2 / : / Ryanodine receptor junctional solenoid repeat / 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. / B30.2/SPRY domain superfamily / Domain in SPla and the RYanodine Receptor. / SPRY domain / Ion transport domain / Ion transport protein / EF-hand domain pair / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / Ryanodine receptor 1
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsNayak, A.R. / Samso, M.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)R01 AR068431 United States
Other privateMDA 352845 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)HSSN261200800001E United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM116789 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24 GM116790 United States
CitationJournal: Elife / Year: 2022
Title: Ca inactivation of the mammalian ryanodine receptor type 1 in a lipidic environment revealed by cryo-EM.
Authors: Ashok R Nayak / Montserrat Samsó /
Abstract: Activation of the intracellular Ca channel ryanodine receptor (RyR) triggers a cytosolic Ca surge, while elevated cytosolic Ca inhibits the channel in a negative feedback mechanism. Cryogenic ...Activation of the intracellular Ca channel ryanodine receptor (RyR) triggers a cytosolic Ca surge, while elevated cytosolic Ca inhibits the channel in a negative feedback mechanism. Cryogenic electron microscopy of rabbit RyR1 embedded in nanodiscs under partially inactivating Ca conditions revealed an open and a closed-inactivated conformation. Ca binding to the high-affinity site engages the central and C-terminal domains into a block, which pries the S6 four-helix bundle open. Further rotation of this block pushes S6 toward the central axis, closing (inactivating) the channel. Main characteristics of the Ca-inactivated conformation are downward conformation of the cytoplasmic assembly and tightly knit subunit interface contributed by a fully occupied Ca activation site, two inter-subunit resolved lipids, and two salt bridges between the EF hand domain and the S2-S3 loop validated by disease-causing mutations. The structural insight illustrates the prior Ca activation prerequisite for Ca inactivation and provides for a seamless transition from inactivated to closed conformations.
History
DepositionDec 31, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 9, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
B: Ryanodine receptor 1,Ryanodine receptor 1,RyR1
C: Ryanodine receptor 1,Ryanodine receptor 1,RyR1
D: Ryanodine receptor 1,Ryanodine receptor 1,RyR1
A: Ryanodine receptor 1,Ryanodine receptor 1,RyR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,137,25620
Polymers2,134,6534
Non-polymers2,60316
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Negative staining
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Ryanodine receptor 1,Ryanodine receptor 1,RyR1 / 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: 533663.250 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / References: UniProt: P11716
#2: Chemical
ChemComp-ACP / PHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER / ADENOSINE-5'-[BETA, GAMMA-METHYLENE]TRIPHOSPHATE


Mass: 505.208 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C11H18N5O12P3 / Comment: AMP-PCP, energy-carrying molecule analogue*YM
#3: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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

ComponentName: Rabbit RyR1 with AMP-PCP and high Ca2+ in nanodisc / Type: COMPLEX
Details: Purified RyR1 was reconstituted with membrane scaffold protein, MSP1E3D1 and POPC.
Entity ID: #1 / Source: NATURAL
Molecular weightValue: 2.26 MDa / Experimental value: YES
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Strain: New Zealand White / Cellular location: Sarcoplasmic Reticulum membrane / Organ: Skeletal Muscle / Organelle: Sarcoplasmic Reticulum
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMMOPS (pH7.4)1
2635 mMPotassium ChlorideKCL1
32 mMDithiothreitol1
42 mMAMP-PCP1
53.7 mMCalcium ChlorideCaCl21
SpecimenConc.: 4.35 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified RyR1 was reconstituted with membrane scaffold protein, MSP1E3D1, and POPC in 1:2:50 molar ratio.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K
Details: Sample was blotted for 1 second on both sides with Whatman hardened ashless filter paper with blot force 2.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 70 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 10002
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 60

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2Latitudeimage acquisition
4Gctf1.06CTF correction
7Coot0.9.5model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
19PHENIXdev-1.20-4444-000model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 311258
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 159444 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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