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- EMDB-6106: Cryo-EM structure of calstabin-bound RYR1-EGTA (class 2) -

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Basic information

Entry
Database: EMDB / ID: EMD-6106
TitleCryo-EM structure of calstabin-bound RYR1-EGTA (class 2)
Map dataReconstruction with C4 symmetry imposed of class 2 of the RYR1-EGTA dataset filtered at 4.8A and sharpened with a B factor of -214
Sample
  • Sample: rabbit skeletal muscle type 1 RyR (RyR1), EGTA-treated, bound to calstabin2 (FKBP12.6)
  • Protein or peptide: Ryanodine Receptor 1
  • Protein or peptide: Peptidyl-prolyl cis-trans isomerase FKBP1B
Keywordscalcium release / excitation-contraction coupling in muscle / closed-state / alpha solenoid scaffold / six-transmembrane ion channel / EF-hands / calstabin / FKBP12 / SPRY
Function / homology
Function and homology information


protein binding / 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 ...protein binding / 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 / intracellularly gated calcium channel activity / outflow tract morphogenesis / monoatomic ion channel activity / toxic substance binding / membrane => GO:0016020 / voltage-gated calcium channel activity / smooth endoplasmic reticulum / striated muscle contraction / skeletal muscle fiber development / monoatomic ion transport / muscle contraction / sarcoplasmic reticulum membrane / release of sequestered calcium ion into cytosol / cellular response to calcium ion / sarcoplasmic reticulum / sarcolemma / calcium ion transmembrane transport / calcium channel activity / transmembrane transport / 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 / 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 ...Ryanodine receptor, SPRY domain 2 / : / Ryanodine receptor junctional solenoid repeat / Ryanodine receptor / 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 / B30.2/SPRY domain profile. / B30.2/SPRY domain superfamily / Domain in SPla and the RYanodine Receptor. / SPRY domain / SPRY domain / Ion transport domain / Ion transport domain / Ion transport protein / EF-hand domain pair / EF-hand domain pair / Concanavalin A-like lectin/glucanase domain superfamily
Similarity search - Domain/homology
Ryanodine receptor 1
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.8 Å
AuthorsZalk R / Clarke OB / des Georges A / Grassucci RA / Reiken S / Mancia F / Hendrickson WA / Frank J / Marks AR
CitationJournal: Nature / Year: 2015
Title: Structure of a mammalian ryanodine receptor.
Authors: Ran Zalk / Oliver B Clarke / Amédée des Georges / Robert A Grassucci / Steven Reiken / Filippo Mancia / Wayne A Hendrickson / Joachim Frank / Andrew R Marks /
Abstract: Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation- ...Ryanodine receptors (RyRs) mediate the rapid release of calcium (Ca(2+)) from intracellular stores into the cytosol, which is essential for numerous cellular functions including excitation-contraction coupling in muscle. Lack of sufficient structural detail has impeded understanding of RyR gating and regulation. Here we report the closed-state structure of the 2.3-megadalton complex of the rabbit skeletal muscle type 1 RyR (RyR1), solved by single-particle electron cryomicroscopy at an overall resolution of 4.8 Å. We fitted a polyalanine-level model to all 3,757 ordered residues in each protomer, defining the transmembrane pore in unprecedented detail and placing all cytosolic domains as tertiary folds. The cytosolic assembly is built on an extended α-solenoid scaffold connecting key regulatory domains to the pore. The RyR1 pore architecture places it in the six-transmembrane ion channel superfamily. A unique domain inserted between the second and third transmembrane helices interacts intimately with paired EF-hands originating from the α-solenoid scaffold, suggesting a mechanism for channel gating by Ca(2+).
History
DepositionSep 29, 2014-
Header (metadata) releaseOct 8, 2014-
Map releaseDec 10, 2014-
UpdateJan 14, 2015-
Current statusJan 14, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6106.gif

Downloads & links

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Map

FileDownload / File: emd_6106.map.gz / Format: CCP4 / Size: 117.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction with C4 symmetry imposed of class 2 of the RYR1-EGTA dataset filtered at 4.8A and sharpened with a B factor of -214
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.66 Å/pix.
x 316 pix.
= 524.56 Å		1.66 Å/pix.
x 316 pix.
= 524.56 Å		1.66 Å/pix.
x 316 pix.
= 524.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.66 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.05748941 - 0.10936084
Average (Standard dev.)-0.00003626 (±0.00499099)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions316316316
Spacing316316316
CellA=B=C: 524.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.661.661.66
M x/y/z316316316
origin x/y/z0.0000.0000.000
length x/y/z524.560524.560524.560
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS316316316
D min/max/mean-0.0570.109-0.000

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Supplemental data

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Supplemental map: emd 6106 additional 1.map

Fileemd_6106_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : rabbit skeletal muscle type 1 RyR (RyR1), EGTA-treated, bound to ...

EntireName: rabbit skeletal muscle type 1 RyR (RyR1), EGTA-treated, bound to calstabin2 (FKBP12.6)
Components
  • Sample: rabbit skeletal muscle type 1 RyR (RyR1), EGTA-treated, bound to calstabin2 (FKBP12.6)
  • Protein or peptide: Ryanodine Receptor 1
  • Protein or peptide: Peptidyl-prolyl cis-trans isomerase FKBP1B

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Supramolecule #1000: rabbit skeletal muscle type 1 RyR (RyR1), EGTA-treated, bound to ...

SupramoleculeName: rabbit skeletal muscle type 1 RyR (RyR1), EGTA-treated, bound to calstabin2 (FKBP12.6)
type: sample / ID: 1000 / Oligomeric state: heterooctamer / Number unique components: 2
Molecular weightTheoretical: 3 MDa / Method: sequence

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Macromolecule #1: Ryanodine Receptor 1

MacromoleculeName: Ryanodine Receptor 1 / type: protein_or_peptide / ID: 1 / Name.synonym: RYR1 / Details: Closed state, EGTA treated / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Tissue: skeletal muscle
Molecular weightExperimental: 2.2 MDa / Theoretical: 2.2 MDa
SequenceUniProtKB: Ryanodine receptor 1
GO: intracellular calcium ion homeostasis, ryanodine-sensitive calcium-release channel activity, membrane => GO:0016020, monoatomic ion transport, transmembrane transport, calcium ion transmembrane ...GO: intracellular calcium ion homeostasis, ryanodine-sensitive calcium-release channel activity, membrane => GO:0016020, monoatomic ion transport, transmembrane transport, calcium ion transmembrane transport, monoatomic ion channel activity, calcium channel activity, calcium ion binding, protein binding
InterPro: Ryanodine receptor, SPRY domain, B30.2/SPRY domain, EF-hand domain pair, Ion transport domain

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Macromolecule #2: Peptidyl-prolyl cis-trans isomerase FKBP1B

MacromoleculeName: Peptidyl-prolyl cis-trans isomerase FKBP1B / type: protein_or_peptide / ID: 2 / Name.synonym: Calstabin-2, FKBP-12.6 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration8 mg/mL
BufferpH: 7.5
Details: 10 mM HEPES, pH 7.5, 1% w/v CHAPS, 1 M NaCl, 2 mM TCEP, 5 mM EGTA, 0.25% w/v CHAPS, 0.001% w/v DOPC (Avanti)
GridDetails: C-flat CF-1.2/1.3-2C-T, Protochips Inc, NC
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 95 K / Instrument: FEI VITROBOT MARK IV / Method: Wait 30 seconds, blot 1.5 seconds, then plunge.

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Electron microscopy

MicroscopeFEI POLARA 300
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 180,000 times magnification.
Details8 second exposure, 23 frames
DateJul 1, 2014
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 3423 / Average electron dose: 25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 31558 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: -4.0 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 23000
Sample stageSpecimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsMovie frames were aligned with Dosegpu_driftcorr. Particles were picked with Arachnid Autopicker and classified and refined with Relion.
CTF correctionDetails: each particle
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.8 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 46400
Final angle assignmentDetails: 0.5 degree sampling
FSC plot (resolution estimation)

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