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- PDB-6r7m: Tobacco Mosaic Virus (TMV) -

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Basic information

Entry
Database: PDB / ID: 6r7m
TitleTobacco Mosaic Virus (TMV)
ComponentsCapsid protein
KeywordsVIRUS / tobacco / mosaic / TMV / cryo-EM / cryoWriter / microfluidic
Function / homology
Function and homology information


helical viral capsid / structural molecule activity / identical protein binding
Similarity search - Function
Tobacco mosaic virus-like, coat protein / Tobacco mosaic virus-like, coat protein / Tobacco mosaic virus-like, coat protein superfamily / Virus coat protein (TMV like) / Four Helix Bundle (Hemerythrin (Met), subunit A) / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Capsid protein / Capsid protein
Similarity search - Component
Biological speciesTobacco mosaic virus
MethodELECTRON MICROSCOPY / helical 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, 2items
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
DepositionMar 29, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 3, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 17, 2019Group: Data collection / Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.year
Revision 1.2Jul 24, 2019Group: Data collection / Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Jul 31, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.4Nov 27, 2019Group: Derived calculations
Category: pdbx_helical_symmetry / pdbx_struct_assembly ...pdbx_helical_symmetry / pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop / pdbx_struct_oper_list
Revision 1.5May 15, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

Movie
  • Biological unit as representative helical assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-4628
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Capsid protein


Theoretical massNumber of molelcules
Total (without water)17,0921
Polymers17,0921
Non-polymers00
Water00
1
A: Capsid protein
x 40


Theoretical massNumber of molelcules
Total (without water)683,68040
Polymers683,68040
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation39
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 40 / Rise per n subunits: 1.41 Å / Rotation per n subunits: 22.03 °)

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Components

#1: Protein Capsid protein


Mass: 17091.998 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Tobacco mosaic virus / References: UniProt: A0A348G6U1, UniProt: P69687*PLUS

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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: 39.5 MDa / Experimental value: NO
Source (natural)Organism: Tobacco mosaic virus
Details of virusEmpty: NO / Enveloped: NO / Isolate: STRAIN / Type: VIRUS-LIKE PARTICLE
Natural hostOrganism: Nicotiana tabacum
Virus shellName: CP / Diameter: 180 nm
Buffer solutionpH: 7.4
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Details: CryoWriter

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 72 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 523
Image scansWidth: 7676 / Height: 7420 / Movie frames/image: 30 / Used frames/image: 0-30

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Processing

EM software
IDNameVersionCategoryDetails
1RELION3particle selectionbeta
2SerialEM3.6image acquisition
7UCSF Chimera1.13.1model fitting
11RELION3classificationbeta
12RELION33D reconstructionbeta
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 22.03 ° / Axial rise/subunit: 1.41 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 2676
3D reconstructionResolution: 1.92 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52776 / Algorithm: BACK PROJECTION / Symmetry type: HELICAL
Atomic model buildingPDB-ID: 6I5A

6i5a


Accession code: 6I5A / Source name: PDB / Type: experimental model

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