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- EMDB-4628: Cryo-EM structure of Tobacco Mossaic Virus from microfluidic grid... -

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Basic information

Entry
Database: EMDB / ID: EMD-4628
TitleCryo-EM structure of Tobacco Mossaic Virus from microfluidic grid preparation.
Map data
Sample
  • Virus: Tobacco mosaic virus
Function / homologyTobacco mosaic virus-like, coat protein / Tobacco mosaic virus-like, coat protein superfamily / Virus coat protein (TMV like) / helical viral capsid / structural molecule activity / identical protein binding / Capsid protein / Capsid protein
Function and homology information
Biological speciesTobacco mosaic virus
Methodhelical reconstruction / cryo EM / Resolution: 1.92 Å
AuthorsSchmidli C / Albiez S / Rima L / Righetto R / Mohammed I / Oliva P / Kovacik L / Stahlberg H / Braun T
Funding support Switzerland, 2 items
OrganizationGrant numberCountry
Swiss National Science Foundation200021_162521 Switzerland
Swiss National Science Foundation205320_166164 Switzerland
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Microfluidic protein isolation and sample preparation for high-resolution cryo-EM.
Authors: Claudio Schmidli / Stefan Albiez / Luca Rima / Ricardo Righetto / Inayatulla Mohammed / Paolo Oliva / Lubomir Kovacik / Henning Stahlberg / Thomas Braun /
Abstract: High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often ...High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.
History
DepositionFeb 21, 2019-
Header (metadata) releaseJul 3, 2019-
Map releaseJul 3, 2019-
UpdateJul 31, 2019-
Current statusJul 31, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0511
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0511
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6r7m
  • Surface level: 0.0511
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6r7m
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4628.png

Downloads & links

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Map

FileDownload / File: emd_4628.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.812 Å
Density
Contour LevelBy AUTHOR: 0.0511 / Movie #1: 0.0511
Minimum - Maximum-0.17561358 - 0.4090628
Average (Standard dev.)0.00027206176 (±0.009701207)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 341.03998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8120.8120.812
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z341.040341.040341.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.1760.4090.000

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Supplemental data

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Sample components

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Entire : Tobacco mosaic virus

EntireName: Tobacco mosaic virus
Components
  • Virus: Tobacco mosaic virus

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Supramolecule #1: Tobacco mosaic virus

SupramoleculeName: Tobacco mosaic virus / type: virus / ID: 1 / Parent: 0 / NCBI-ID: 12242 / Sci species name: Tobacco mosaic virus / Virus type: VIRUS-LIKE PARTICLE / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Nicotiana tabacum (common tobacco)
Molecular weightTheoretical: 39.5 MDa
Virus shellShell ID: 1 / Name: CP / Diameter: 180.0 Å

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Details: CryoWriter..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 0-30 / Number grids imaged: 1 / Number real images: 523 / Average exposure time: 6.0 sec. / Average electron dose: 72.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Segment selectionNumber selected: 2676 / Software - Name: RELION (ver. 3) / Software - details: beta
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

2842

Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 1.41 Å
Applied symmetry - Helical parameters - Δ&Phi: 22.03 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 1.92 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3) / Software - details: beta / Number images used: 52776
FSC plot (resolution estimation)

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