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- EMDB-22596: Focused cryo-EM map of rabbit RyR1 central and transmembrane doma... -

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Basic information

Entry
Database: EMDB / ID: EMD-22596
TitleFocused cryo-EM map of rabbit RyR1 central and transmembrane domains in the presence of Mg2+ and AMP-PCP in nanodisc
Map dataMasked full map
Sample
  • Complex: Rabbit RyR1 with Mg2+ and AMP-PCP
    • Protein or peptide: RyR1Ryanodine receptor 1
Biological speciesOryctolagus cuniculus (rabbit) / rabbit (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsNayak AR / Samso M
Funding support United States, 6 items
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: To Be Published
Title: Molecular Mechanism of Inhibition of RyR1 by Magnesium
Authors: Nayak AR / Will AH / Hartmann PC / Lobo JJ / Rangubpit W / Sompornpisut P / Samso M
History
DepositionSep 4, 2020-
Header (metadata) releaseSep 22, 2021-
Map releaseSep 22, 2021-
UpdateSep 22, 2021-
Current statusSep 22, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22596.png

Downloads & links

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Map

FileDownload / File: emd_22596.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMasked full map
Voxel sizeX=Y=Z: 1.36 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.04
Minimum - Maximum-0.05851621 - 0.13907781
Average (Standard dev.)0.00030299052 (±0.003540685)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 478.72 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.361.361.36
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z478.720478.720478.720
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-0.0590.1390.000

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Supplemental data

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Mask #1

Fileemd_22596_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unfiltered and unmasked half map1 up to Nyquist frequency

Fileemd_22596_additional_1.map
AnnotationUnfiltered and unmasked half map1 up to Nyquist frequency
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unfiltered and unmasked half map2 up to Nyquist frequency

Fileemd_22596_additional_2.map
AnnotationUnfiltered and unmasked half map2 up to Nyquist frequency
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Masked half map1

Fileemd_22596_half_map_1.map
AnnotationMasked half map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Masked half map2

Fileemd_22596_half_map_2.map
AnnotationMasked half map2
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 RyR1 with Mg2+ and AMP-PCP

EntireName: Rabbit RyR1 with Mg2+ and AMP-PCP
Components
  • Complex: Rabbit RyR1 with Mg2+ and AMP-PCP
    • Protein or peptide: RyR1Ryanodine receptor 1

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Supramolecule #1: Rabbit RyR1 with Mg2+ and AMP-PCP

SupramoleculeName: Rabbit RyR1 with Mg2+ and AMP-PCP / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Purified RyR1 was reconstituted with membrane scaffold protein, MSP1E3D1, and POPC.
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Strain: New Zealand White / Organ: Skeletal Muscle / Organelle: Sarcoplasmic Reticulum / Location in cell: Sarcoplasmic Reticulum membrane
Molecular weightExperimental: 2.26 MDa

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Macromolecule #1: RyR1

MacromoleculeName: RyR1 / type: protein_or_peptide / ID: 1
Details: Rabbit RyR1 corresponding to amino acid sequence 3668-5037
Enantiomer: LEVO
Source (natural)Organism: rabbit (rabbit) / Strain: New Zealand White / Organ: skeletal muscle
SequenceString: SFEDRMIDDL SKAGEQEEEE EEVEEKKPDP LHQLVLHFSR TALTEKSKLD EDYLYMAYAD IMAKSCHLEE GGENEEVEV SFEEKEMEKQ RLLYQQSRLH TRGAAEMVLQ MISACKGETG AMVSSTLKLG ISILNGGNAE V QQKMLDYL KDKKEVGFFQ SIQALMQTCS ...String:
SFEDRMIDDL SKAGEQEEEE EEVEEKKPDP LHQLVLHFSR TALTEKSKLD EDYLYMAYAD IMAKSCHLEE GGENEEVEV SFEEKEMEKQ RLLYQQSRLH TRGAAEMVLQ MISACKGETG AMVSSTLKLG ISILNGGNAE V QQKMLDYL KDKKEVGFFQ SIQALMQTCS VLDLNAFERQ NKAEGLGMVN EDGTVINRQN GEKVMADDEF TQ DLFRFLQ LLCEGHNNDF QNYLRTQTGN TTTINIIICT VDYLLRLQES ISDFYWYYSG KDVIEEQGKR NFS KAMSVA KQVFNSLTEY IQGPCTGNQQ SLAHSRLWDA VVGFLHVFAH MMMKLAQDSS QIELLKELLD LQKD MVVML LSLLEGNVVN GMIARQMVDM LVESSSNVEM ILKFFDMFLK LKDIVGSEAF QDYVTDPRGL ISKKD FQKA MDSQKQFTGP EIQFLLSCSE ADENEMINFE EFANRFQEPA RDIGFNVAVL LTNLSEHV P HDPRLRNFLE LAESILEYFR PYLGRAEIMG ASRRIERIYF EISETNRAQW EMPQVKESKR QFIFDVVNE GGEAEKMELF VSFCEDTIFE MQIAAQISEF WGELEVQRVK FLNYLSRNFY TLRFLALFLA FAINFILLF YKVSDSPPAN MVYYFLEEST GYMEPALWCL SLLHTLVAFL CIIGYNCLKV PLVIFKREKE LARKLEFDGL YITEQPGDD DVKGQWDRLV LNTPSFPSNY WDKFVKRKVL DKHGDIFGRE RIAELLGMDL ASLEITAHNE R KPDPPPGL LTWLMSIDVK YQIWKFGVIF TDNSFLYLGW YMVMSLLGHY NNFFFAAHLL DIAMGVKTLR TI LSSVTHN GKQLVMTVGL LAVVVYLYTV VAFNFFRKFY NKSEDEDEPD MKCDDMMTCY LFHMYVGVRA GGG IGDEIE DPAGDEYELY RVVFDITFFF FVIVILLAII QGLIIDAFGE LRDQQEQVKE DMETKCFICG IGSD YFDTT PHGFETHTLE EHNLANYMFF LMYLINKDET EHTGQESYVW KMYQERCWDF FPAGDCFRKQ YEDQL S

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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

Concentration4.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationNameFormula
20.0 mMMOPS (pH7.4)
635.0 mMPotassium ChlorideKCl
2.0 mMDithiothreitol
5.0 mMAMP-PCP
5.0 mMMagnesium ChlorideMgCl2
2.0 mMEGTA
GridModel: C-flat-2/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: Sample was blotted for 1 second on both sides with Whatman hardened ashless filter paper with blot force 2..
DetailsPurified RyR1 was reconstituted with membrane scaffold protein, MSP1E3D1, and POPC in 1:2:50 molar ratio.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 3371 / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 115741
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 68155
FSC plot (resolution estimation)

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