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- EMDB-21384: Map of an acid-sensing ion channel in a desensitized state after ... -

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Basic information

Entry
Database: EMDB / ID: EMD-21384
TitleMap of an acid-sensing ion channel in a desensitized state after photo-uncaging protons.
Map dataFull map of an acid-sensing ion channel in a desensitized state after photo-uncaging protons.
Sample
  • Complex: Acid-sensing ion channel
    • Protein or peptide: Acid-sensing ion channel
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsYoder N / Jalali-Yazdi F / Gouaux E
Funding support United States, 4 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)5T32DK007680 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)5F31NS096782 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)5R01NS038631 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)5F32MH115595 United States
CitationJournal: J Struct Biol / Year: 2020
Title: Light-coupled cryo-plunger for time-resolved cryo-EM.
Authors: Nate Yoder / Farzad Jalali-Yazdi / Sigrid Noreng / Alexandra Houser / Isabelle Baconguis / Eric Gouaux /
Abstract: Proteins are dynamic molecules that can undergo rapid conformational rearrangements in response to stimuli. These structural changes are often critical to protein function, and thus elucidating time- ...Proteins are dynamic molecules that can undergo rapid conformational rearrangements in response to stimuli. These structural changes are often critical to protein function, and thus elucidating time-dependent conformational landscapes has been a long-standing goal of structural biology. To harness the power of single particle cryo-EM methods to enable 'time-resolved' structure determination, we have developed a light-coupled cryo-plunger that pairs flash-photolysis of caged ligands with rapid sample vitrification. The 'flash-plunger' consists of a high-power ultraviolet LED coupled with focusing optics and a motorized linear actuator, enabling the user to immobilize protein targets in vitreous ice within a programmable time window - as short as tens of milliseconds - after stimulus delivery. The flash-plunger is a simple, inexpensive and flexible tool to explore short-lived conformational states previously unobtainable by conventional sample preparation methods.
History
DepositionFeb 13, 2020-
Header (metadata) releaseOct 7, 2020-
Map releaseOct 7, 2020-
UpdateOct 21, 2020-
Current statusOct 21, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.18
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.18
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21384.png

Downloads & links

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Map

FileDownload / File: emd_21384.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map of an acid-sensing ion channel in a desensitized state after photo-uncaging protons.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 332. Å		0.83 Å/pix.
x 400 pix.
= 332. Å		0.83 Å/pix.
x 400 pix.
= 332. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18 / Movie #1: 0.18
Minimum - Maximum-0.58375794 - 1.6548045
Average (Standard dev.)-0.0014565802 (±0.03140866)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 332.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.830.830.83
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z332.000332.000332.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.5841.655-0.001

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Supplemental data

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Additional map: A map of the extracellular domain of an...

Fileemd_21384_additional_1.map
AnnotationA map of the extracellular domain of an acid-sensing ion channel in a proton-bound conformation after photo-uncaging protons.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Acid-sensing ion channel

EntireName: Acid-sensing ion channel
Components
  • Complex: Acid-sensing ion channel
    • Protein or peptide: Acid-sensing ion channel

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Supramolecule #1: Acid-sensing ion channel

SupramoleculeName: Acid-sensing ion channel / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Map of the entire channel.
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 180 KDa

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Macromolecule #1: Acid-sensing ion channel

MacromoleculeName: Acid-sensing ion channel / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MMDLKVDEEE VDSGQPVSIQ AFASSSTLHG ISHIFSYERL SLKRVVWALC FMGSLALLAL VCTNRIQYY FLYPHVTKLD EVAATRLTFP AVTFCNLNEF RFSRVTKNDL YHAGELLALL N NRYEIPDT QTADEKQLEI LQDKANFRNF KPKPFNMLEF YDRAGHDIRE ...String:
MMDLKVDEEE VDSGQPVSIQ AFASSSTLHG ISHIFSYERL SLKRVVWALC FMGSLALLAL VCTNRIQYY FLYPHVTKLD EVAATRLTFP AVTFCNLNEF RFSRVTKNDL YHAGELLALL N NRYEIPDT QTADEKQLEI LQDKANFRNF KPKPFNMLEF YDRAGHDIRE MLLSCFFRGE QC SPEDFKV VFTRYGKCYT FNAGQDGKPR LITMKGGTGN GLEIMLDIQQ DEYLPVWGET DET SFEAGI KVQIHSQDEP PLIDQLGFGV APGFQTFVSC QEQRLIYLPP PWGDCKATTG DSEF YDTYS ITACRIDCET RYLVENCNCR MVHMPGDAPY CTPEQYKECA DPALDFLVEK DNEYC VCEM PCNVTRYGKE LSMVKIPSKA SAKYLAKKYN KSEQYIGENI LVLDIFFEAL NYETIE QKK AYEVAGLLGD IGGQMGLFIG ASILTVLELF DYAYEVIKHR LCRRGKCRKN HKRNNTD KG VALSMDDVKR HNPCESLRGH PAGMTYAANI LPHHPARGTF EDFTC

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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

Concentration1.00 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/1 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 70 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: Specimen was frozen on a custom-made manual plunge apparatus in a cold room environment maintained at high humidity. Vitrification occurred following 25 ms of exposure to high power UV irradiation..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing #1

Image processing ID1
CTF correctionSoftware - Name: Gctf
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2) / Number images used: 45135
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)

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Image processing #2

Image processing ID2
CTF correctionSoftware - Name: Gctf
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2)
Details: Final statistics associated with local refinement of extracellular domain region.
Number images used: 45135
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2)
Details: Local refinement done on region of the map corresponding to the less-flexible extracellular domain. Particle subtraction done to removed transmembrane region. All data processing was performed in cryoSPARC V2.

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