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- PDB-7k0t: Cryo-EM structure of rabbit RyR1 in the presence of AMP-PCP in na... -

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Basic information

Entry
Database: PDB / ID: 7k0t
TitleCryo-EM structure of rabbit RyR1 in the presence of AMP-PCP in nanodisc
ComponentsRyR1
KeywordsTRANSPORT PROTEIN / Ryanodine Receptor / RyR1 / Intracellular Calcium channel / Excitation-Contraction coupling / ATP
Function / homologyPHOSPHOMETHYLPHOSPHONIC ACID ADENYLATE ESTER
Function and homology information
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsNayak, A.R. / Samso, M.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIH/NIAMS)R01 AR068431 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01 HL133182 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
DepositionSep 5, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 22, 2021Provider: repository / Type: Initial release
Revision 2.0Mar 9, 2022Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Database references / Derived calculations / Polymer sequence / Source and taxonomy / Structure summary
Category: atom_site / em_entity_assembly ...atom_site / em_entity_assembly / em_software / entity / entity_poly / entity_poly_seq / entity_src_nat / pdbx_contact_author / pdbx_database_related / pdbx_entity_instance_feature / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_poly_seq_scheme / pdbx_struct_conn_angle / pdbx_struct_sheet_hbond / pdbx_unobs_or_zero_occ_atoms / pdbx_unobs_or_zero_occ_residues / pdbx_validate_close_contact / pdbx_validate_main_chain_plane / pdbx_validate_peptide_omega / pdbx_validate_polymer_linkage / pdbx_validate_rmsd_angle / pdbx_validate_rmsd_bond / pdbx_validate_torsion / struct_asym / struct_conf / struct_conn / struct_ref / struct_ref_seq / struct_sheet_range
Item: _em_entity_assembly.details / _em_entity_assembly.entity_id_list ..._em_entity_assembly.details / _em_entity_assembly.entity_id_list / _em_software.category / _pdbx_entity_nonpoly.entity_id / _pdbx_nonpoly_scheme.auth_seq_num / _pdbx_nonpoly_scheme.entity_id / _pdbx_nonpoly_scheme.pdb_strand_id / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_sheet_hbond.range_1_auth_asym_id / _pdbx_struct_sheet_hbond.range_1_auth_seq_id / _pdbx_struct_sheet_hbond.range_1_label_seq_id / _pdbx_struct_sheet_hbond.range_2_auth_asym_id / _pdbx_struct_sheet_hbond.range_2_auth_seq_id / _pdbx_struct_sheet_hbond.range_2_label_seq_id / _struct_asym.entity_id / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_ref_seq.db_align_beg / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_strand_id / _struct_ref_seq.ref_id / _struct_ref_seq.seq_align_end / _struct_sheet_range.beg_auth_asym_id / _struct_sheet_range.beg_auth_seq_id / _struct_sheet_range.beg_label_seq_id / _struct_sheet_range.end_auth_asym_id / _struct_sheet_range.end_auth_seq_id / _struct_sheet_range.end_label_seq_id
Description: Polymer backbone linkage / Provider: author / Type: Coordinate replacement
Revision 2.1Mar 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
Revision 2.2May 29, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

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Assembly

Deposited unit
A: RyR1
B: RyR1
D: RyR1
C: RyR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)2,136,93512
Polymers2,134,6534
Non-polymers2,2828
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
RyR1


Mass: 533663.250 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / Organ: Skeletal Muscle / Strain: New Zealand White
#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 / Feature type: SUBJECT OF INVESTIGATION / 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
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 / 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
45 mMAMP-PCP1
51 mMEGTA1
61 mMEDTA1
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: UltrAuFoil 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: 130000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 70.3 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1959
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 60

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Processing

EM software
IDNameVersionCategory
2Latitudeimage acquisition
4Gctf1.06CTF correction
7Coot0.8.9.1model fitting
9PHENIXdev-3958-000model refinement
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 52856
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21551 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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