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- PDB-7q2r: cryo iDPC-STEM structure recorded with CSA 4.0 -

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Basic information

Entry
Database: PDB / ID: 7q2r
Titlecryo iDPC-STEM structure recorded with CSA 4.0
Components
  • Capsid proteinCapsid
  • RNA (5'-R(P*GP*AP*A)-3')
KeywordsVIRUS / tobacco mosaic virus / RNA 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 / RNA / Capsid protein
Function and homology information
Biological speciesTobacco mosaic virus
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsSachse, C. / Leidl, M.L.
Funding support Germany, 1items
OrganizationGrant numberCountry
Helmholtz Association Germany
CitationJournal: Nat Methods / Year: 2022
Title: Single-particle cryo-EM structures from iDPC-STEM at near-atomic resolution.
Authors: Ivan Lazić / Maarten Wirix / Max Leo Leidl / Felix de Haas / Daniel Mann / Maximilian Beckers / Evgeniya V Pechnikova / Knut Müller-Caspary / Ricardo Egoavil / Eric G T Bosch / Carsten Sachse /
Abstract: In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently ...In electron cryomicroscopy (cryo-EM), molecular images of vitrified biological samples are obtained by conventional transmission microscopy (CTEM) using large underfocuses and subsequently computationally combined into a high-resolution three-dimensional structure. Here, we apply scanning transmission electron microscopy (STEM) using the integrated differential phase contrast mode also known as iDPC-STEM to two cryo-EM test specimens, keyhole limpet hemocyanin (KLH) and tobacco mosaic virus (TMV). The micrographs show complete contrast transfer to high resolution and enable the cryo-EM structure determination for KLH at 6.5 Å resolution, as well as for TMV at 3.5 Å resolution using single-particle reconstruction methods, which share identical features with maps obtained by CTEM of a previously acquired same-sized TMV data set. These data show that STEM imaging in general, and in particular the iDPC-STEM approach, can be applied to vitrified single-particle specimens to determine near-atomic resolution cryo-EM structures of biological macromolecules.
History
DepositionOct 26, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Capsid protein
B: RNA (5'-R(P*GP*AP*A)-3')


Theoretical massNumber of molelcules
Total (without water)18,0512
Polymers18,0512
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area750 Å2
ΔGint4 kcal/mol
Surface area9600 Å2

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Components

#1: Protein Capsid protein / Capsid / Coat protein


Mass: 17091.998 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tobacco mosaic virus (vulgare) / Strain: vulgare / References: UniProt: P69687
#2: RNA chain RNA (5'-R(P*GP*AP*A)-3')


Mass: 958.660 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tobacco mosaic virus (vulgare)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: Tobacco mosaic virus / Type: VIRUS / Entity ID: all / Source: NATURAL
Molecular weightValue: 131 kDa/nm / Experimental value: NO
Source (natural)Organism: Tobacco mosaic virus
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Tobacco mosaic virus
Buffer solutionpH: 7.4
SpecimenConc.: 90 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: blot force of +10 and a duration for blotting of 10 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: iDPC-STEM
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm
Image recordingElectron dose: 35 e/Å2 / Film or detector model: OTHER

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Processing

EM software
IDNameVersionCategory
1EMAN22particle selection
7UCSF Chimeramodel fitting
13RELION3.13D reconstruction
Image processingDetails: iDPC-STEM
CTF correctionType: NONE
Helical symmertyAngular rotation/subunit: 22.03 ° / Axial rise/subunit: 1.408 Å / Axial symmetry: C1
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 2224 / Symmetry type: HELICAL
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 4UDV

4udv

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