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- EMDB-1606: RyR1-FKBP12 in the closed conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-1606
TitleRyR1-FKBP12 in the closed conformation
Map data3D reconstruction of RyR1 in the closed conformation.
Sample
  • Sample: ryanodine receptor 1 (RyR1) with FKBP12 bound
  • Protein or peptide: Ryanodine receptor isoform 1
Keywordsryanodine receptor / FKBP12 / channel gating
Function / homologyryanodine-sensitive calcium-release channel activity / RIH domain
Function and homology information
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.2 Å
AuthorsSamso M / Feng W / Pessah IN / Allen PD
CitationJournal: PLoS Biol / Year: 2009
Title: Coordinated movement of cytoplasmic and transmembrane domains of RyR1 upon gating.
Authors: Montserrat Samsó / Wei Feng / Isaac N Pessah / P D Allen /
Abstract: Ryanodine receptor type 1 (RyR1) produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how ...Ryanodine receptor type 1 (RyR1) produces spatially and temporally defined Ca2+ signals in several cell types. How signals received in the cytoplasmic domain are transmitted to the ion gate and how the channel gates are unknown. We used EGTA or neuroactive PCB 95 to stabilize the full closed or open states of RyR1. Single-channel measurements in the presence of FKBP12 indicate that PCB 95 inverts the thermodynamic stability of RyR1 and locks it in a long-lived open state whose unitary current is indistinguishable from the native open state. We analyzed two datasets of 15,625 and 18,527 frozen-hydrated RyR1-FKBP12 particles in the closed and open conformations, respectively, by cryo-electron microscopy. Their corresponding three-dimensional structures at 10.2 A resolution refine the structure surrounding the ion pathway previously identified in the closed conformation: two right-handed bundles emerging from the putative ion gate (the cytoplasmic "inner branches" and the transmembrane "inner helices"). Furthermore, six of the identifiable transmembrane segments of RyR1 have similar organization to those of the mammalian Kv1.2 potassium channel. Upon gating, the distal cytoplasmic domains move towards the transmembrane domain while the central cytoplasmic domains move away from it, and also away from the 4-fold axis. Along the ion pathway, precise relocation of the inner helices and inner branches results in an approximately 4 A diameter increase of the ion gate. Whereas the inner helices of the K+ channels and of the RyR1 channel cross-correlate best with their corresponding open/closed states, the cytoplasmic inner branches, which are not observed in the K+ channels, appear to have at least as important a role as the inner helices for RyR1 gating. We propose a theoretical model whereby the inner helices, the inner branches, and the h1 densities together create an efficient novel gating mechanism for channel opening by relaxing two right-handed bundle structures along a common 4-fold axis.
History
DepositionMar 19, 2009-
Header (metadata) releaseApr 15, 2009-
Map releaseMar 25, 2011-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.95
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 1.95
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1606.png

Downloads & links

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Map

FileDownload / File: emd_1606.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of RyR1 in the closed conformation.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.8 Å/pix.
x 180 pix.
= 504. Å		2.8 Å/pix.
x 180 pix.
= 504. Å		2.8 Å/pix.
x 180 pix.
= 504. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.95 / Movie #1: 1.95
Minimum - Maximum-1.04341567 - 3.84153962
Average (Standard dev.)-0.13235998 (±0.45409393)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 504.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z504.000504.000504.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-17-17-200
NX/NY/NZ123123401
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-1.0433.842-0.132

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Supplemental data

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Sample components

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Entire : ryanodine receptor 1 (RyR1) with FKBP12 bound

EntireName: ryanodine receptor 1 (RyR1) with FKBP12 bound
Components
  • Sample: ryanodine receptor 1 (RyR1) with FKBP12 bound
  • Protein or peptide: Ryanodine receptor isoform 1

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Supramolecule #1000: ryanodine receptor 1 (RyR1) with FKBP12 bound

SupramoleculeName: ryanodine receptor 1 (RyR1) with FKBP12 bound / type: sample / ID: 1000
Oligomeric state: RyR1 forms a homotetramer and one FKBP12 binds to each RyR1 subunit
Number unique components: 2
Molecular weightTheoretical: 2.26 MDa

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Macromolecule #1: Ryanodine receptor isoform 1

MacromoleculeName: Ryanodine receptor isoform 1 / type: protein_or_peptide / ID: 1 / Name.synonym: RyR1 / Number of copies: 4 / Oligomeric state: Tetramer / Recombinant expression: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Tissue: Fast twitch skeletal muscle / Organelle: Sarcoplasmic reticulum / Location in cell: Sarcoplasmic reticulum membrane
Molecular weightTheoretical: 2.26 MDa
SequenceGO: ryanodine-sensitive calcium-release channel activity / InterPro: RIH domain

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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

Concentration2.00 mg/mL
BufferpH: 7.4
Details: 20 mM Na-MOPS pH 7.4, 0.9 M NaCl, 0.5% (w/v) CHAPS, 2 mM DTT, 2 mM EGTA
GridDetails: 300 mesh holey copper grids
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 77 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: two-side blotting plunger
Method: Blot for 4 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.8 µm / Nominal defocus min: 2.3 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 87 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 150,000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7.0 µm / Number real images: 257 / Average electron dose: 9 e/Å2 / Details: After scanning pixels were averaged 2x2 / Bits/pixel: 8
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: FREALIGN SPIDER / Number images used: 9331

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Atomic model buiding 1

Initial modelPDB ID:

1bl8

RefinementSpace: REAL

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Atomic model buiding 2

Initial modelPDB ID:

1p7b

RefinementSpace: REAL

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