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- PDB-4udv: Cryo-EM structure of TMV at 3.35 A resolution -

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Basic information

Entry
Database: PDB / ID: 4udv
TitleCryo-EM structure of TMV at 3.35 A resolution
Components
  • 5'-D(*GP*AP*AP)-3'
  • CAPSID PROTEIN
KeywordsVIRAL PROTEIN / DIRECT ELECTRON DETECTORS / SINGLE PARTICLE HELICAL RECONSTRUCTION / HIGH RESOLUTION
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
RNA / Capsid protein
Similarity search - Component
Biological speciesTOBACCO MOSAIC VIRUS
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.35 Å
AuthorsFromm, S.A. / Bharat, T.A.M. / Jakobi, A.J. / Hagen, W.J.H. / Sachse, C.
CitationJournal: J Struct Biol / Year: 2015
Title: Seeing tobacco mosaic virus through direct electron detectors.
Authors: Simon A Fromm / Tanmay A M Bharat / Arjen J Jakobi / Wim J H Hagen / Carsten Sachse /
Abstract: With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require ...With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.
History
DepositionDec 11, 2014Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 31, 2014Provider: repository / Type: Initial release
Revision 1.1Feb 18, 2015Group: Database references
Revision 1.2Oct 3, 2018Group: Author supporting evidence / Data collection / Structure summary
Category: diffrn_radiation / diffrn_radiation_wavelength ...diffrn_radiation / diffrn_radiation_wavelength / em_single_particle_entity / em_software / entity
Item: _em_software.image_processing_id / _entity.src_method
Revision 1.3May 8, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Biological unit as representative helical assembly
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-2842
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  • Superimposition on EM map
  • EMDB-2842
  • Imaged by UCSF Chimera
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  • Superimposition on EM map
  • EMDB-2842
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Structure viewerMolecule:
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Assembly

Deposited unit
A: CAPSID PROTEIN
R: 5'-D(*GP*AP*AP)-3'


Theoretical massNumber of molelcules
Total (without water)18,4642
Polymers18,4642
Non-polymers00
Water00
1
A: CAPSID PROTEIN
R: 5'-D(*GP*AP*AP)-3'
x 49


Theoretical massNumber of molelcules
Total (without water)904,74098
Polymers904,74098
Non-polymers00
Water0
TypeNameSymmetry operationNumber
helical symmetry operation48
identity operation1_555x,y,z1
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit
  • helical asymmetric unit, std helical frame
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 49 / Rise per n subunits: 1.408 Å / Rotation per n subunits: 22.03 °)

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Components

#1: Protein CAPSID PROTEIN / COAT PROTEIN / CP


Mass: 17505.426 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) TOBACCO MOSAIC VIRUS / Strain: VULGARE / References: UniProt: P69687
#2: RNA chain 5'-D(*GP*AP*AP)-3'


Mass: 958.660 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) TOBACCO MOSAIC VIRUS / Strain: VULGARE

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: TOBACCO MOSAIC VIRUS / Type: VIRUS
Buffer solutionName: 50 MM NAPO4 / pH: 7 / Details: 50 MM NAPO4
SpecimenConc.: 11 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE
Details: CRYOGEN - ETHANE, HUMIDITY - 90 PERCENT, TEMPERATURE - 95 K, INSTRUMENT - FEI VITROBOT MARK III PROCEDURE - BLOT FOR 8 SECONDS WITH AN OFFSET OF -2 MM CA 30-45 SECONDS AFTER SAMPLE APPLICATION

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Oct 16, 2013
Details: NANOPROBE MODE, DOSE RATE CA 41 E- PX S ON THE CAMERA LEVEL, FULLY AUTOMATED ACQUISITION USING FEI EPU SOFTWARE
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 131827 X / Nominal defocus max: 4500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 30.7 e/Å2 / Film or detector model: FEI FALCON II (4k x 4k)
Image scansNum. digital images: 109

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Processing

EM softwareName: SPRING / Category: 3D reconstruction
CTF correctionDetails: CTFTILT (SPECIFIC FOR EACH SEGMENT)
3D reconstructionMethod: PROJECTION MATCHING / Resolution: 3.35 Å / Num. of particles: 450000 / Nominal pixel size: 1.062 Å / Actual pixel size: 1.062 Å / Magnification calibration: LAYER LINE CORRELATION
Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-2842. (DEPOSITION ID: 12988).
Symmetry type: HELICAL
Atomic model buildingB value: 90 / Protocol: OTHER / Space: REAL / Target criteria: REAL SPACE CORRELATION
Details: METHOD--LOCAL AND GLOBAL CORRELATION REFINEMENT PROTOCOL--FIBRE DIFFRACTION
RefinementHighest resolution: 3.35 Å
Refinement stepCycle: LAST / Highest resolution: 3.35 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1206 67 0 0 1273

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