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- PDB-1m0f: Structural Studies of Bacteriophage alpha3 Assembly, Cryo-electro... -

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Basic information

Entry
Database: PDB / ID: 1m0f
TitleStructural Studies of Bacteriophage alpha3 Assembly, Cryo-electron microscopy
Components
  • Capsid Protein F
  • Major Spike Protein G
  • Scaffolding Protein B
  • Scaffolding protein D
KeywordsVIRUS / Bacteriophage / Cryo Electron Microscopy / Procapsid / morphogenesis / microviridae / assembly / Icosahedral virus
Function / homology
Function and homology information


modulation by virus of host process / viral procapsid maturation / T=1 icosahedral viral capsid / viral capsid / host cell cytoplasm / symbiont entry into host cell / virion attachment to host cell / structural molecule activity
Similarity search - Function
Scaffold protein D / Scaffold protein D superfamily / Bacteriophage scaffolding protein D / Major spike protein G / Major spike protein (G protein) / Microviridae F protein / Microviridae F protein superfamily / Capsid protein (F protein) / Capsid/spike protein, ssDNA virus / Viral coat protein subunit
Similarity search - Domain/homology
Capsid protein F / Major spike protein G / External scaffolding protein D
Similarity search - Component
Biological speciesEnterobacteria phage alpha3 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 16 Å
AuthorsBernal, R.A. / Hafenstein, S. / Olson, N.H. / Bowman, V.D. / Chipman, P.R. / Baker, T.S. / Fane, B.A. / Rossmann, M.G.
CitationJournal: J Mol Biol / Year: 2003
Title: Structural studies of bacteriophage alpha3 assembly.
Authors: Ricardo A Bernal / Susan Hafenstein / Norman H Olson / Valorie D Bowman / Paul R Chipman / Timothy S Baker / Bentley A Fane / Michael G Rossmann /
Abstract: Bacteriophage alpha3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include phiX174. These viruses have an ssDNA genome associated with approximately 12 ...Bacteriophage alpha3 is a member of the Microviridae, a family of small, single-stranded, icosahedral phages that include phiX174. These viruses have an ssDNA genome associated with approximately 12 copies of an H pilot protein and 60 copies of a small J DNA-binding protein. The surrounding capsid consists of 60 F coat proteins decorated with 12 pentameric spikes of G protein. Assembly proceeds via a 108S empty procapsid that requires the external D and internal B scaffolding proteins for its formation. The alpha3 "open" procapsid structural intermediate was determined to 15A resolution by cryo-electron microscopy (cryo-EM). Unlike the phiX174 "closed" procapsid and the infectious virion, the alpha3 open procapsid has 30A wide pores at the 3-fold vertices and 20A wide gaps between F pentamers as a result of the disordering of two helices in the F capsid protein. The large pores are probably used for DNA entry and internal scaffolding protein exit during DNA packaging. Portions of the B scaffolding protein are located at the 5-fold axes under the spike and in the hydrophobic pocket on the inner surface of the capsid. Protein B appears to have autoproteolytic activity that cleaves at an Arg-Phe motif and probably facilitates the removal of the protein through the 30A wide pores. The structure of the alpha3 mature virion was solved to 3.5A resolution by X-ray crystallography and was used to interpret the open procapsid cryo-EM structure. The main differences between the alpha3 and phiX174 virion structures are in the spike and the DNA-binding proteins. The alpha3 pentameric spikes have a rotation of 3.5 degrees compared to those of phiX174. The alpha3 DNA-binding protein, which is shorter by 13 amino acid residues at its amino end when compared to the phiX174 J protein, retains its carboxy-terminal-binding site on the internal surface of the capsid protein. The icosahedrally ordered structural component of the ssDNA appears to be substantially increased in alpha3 compared to phiX174, allowing the building of about 10% of the ribose-phosphate backbone.
History
DepositionJun 12, 2002Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 25, 2002Provider: repository / Type: Initial release
Revision 1.1Apr 28, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Version format compliance
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Feb 14, 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
Revision 1.5Apr 17, 2024Group: Other / Category: pdbx_database_status / Item: _pdbx_database_status.status_code_sf
Remark 999SEQUENCE Residue 160 of the F protein is an ARG according to the reported sequence but no density ...SEQUENCE Residue 160 of the F protein is an ARG according to the reported sequence but no density is seen for the side chain in the crystal structure for this residue. After a structural sequence alignment with homologous bacteriophages phix174 and G4, residue 160 was found to be a glycine in the other phages. Consequently, the authors state residue 160 should be a Glycine. CHAINS 1,2,3,4 ARE SCAFFOLDING PROTEIN D AND CHAIN B IS SCAFFOLDING PROTEIN B, ALL FROM BACTERIOPHAGE ALPHA3. THE PROTEIN SEQUENCES WERE TAKEN FROM 1CD3 PHIX174 COORDINATES AND FITTED INTO THE CRYO-EM STRUCTURE. THE SEQUENCE DATABASE REFERENCE FOR THE PHIX174 SCAFFOLDING PROTEINS D AND B ARE P03637 AND P07929 RESPECTIVELY.

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
1: Scaffolding protein D
2: Scaffolding protein D
3: Scaffolding protein D
4: Scaffolding protein D
F: Capsid Protein F
G: Major Spike Protein G
B: Scaffolding Protein B


Theoretical massNumber of molelcules
Total (without water)144,8577
Polymers144,8577
Non-polymers00
Water0
1
1: Scaffolding protein D
2: Scaffolding protein D
3: Scaffolding protein D
4: Scaffolding protein D
F: Capsid Protein F
G: Major Spike Protein G
B: Scaffolding Protein B
x 60


Theoretical massNumber of molelcules
Total (without water)8,691,394420
Polymers8,691,394420
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
1: Scaffolding protein D
2: Scaffolding protein D
3: Scaffolding protein D
4: Scaffolding protein D
F: Capsid Protein F
G: Major Spike Protein G
B: Scaffolding Protein B
x 5


  • icosahedral pentamer
  • 724 kDa, 35 polymers
Theoretical massNumber of molelcules
Total (without water)724,28335
Polymers724,28335
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
1: Scaffolding protein D
2: Scaffolding protein D
3: Scaffolding protein D
4: Scaffolding protein D
F: Capsid Protein F
G: Major Spike Protein G
B: Scaffolding Protein B
x 6


  • icosahedral 23 hexamer
  • 869 kDa, 42 polymers
Theoretical massNumber of molelcules
Total (without water)869,13942
Polymers869,13942
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Hermann–Mauguin notation: 532 / Schoenflies symbol: I (icosahedral))

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Components

#1: Protein
Scaffolding protein D / / GPD / Coordinate model: Cα atoms only


Mass: 16953.316 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage alpha3 (virus) / Genus: Microvirus / Production host: Escherichia coli (E. coli) / References: UniProt: P69486*PLUS
#2: Protein Capsid Protein F / / F protein / GPF / Coordinate model: Cα atoms only


Mass: 49294.277 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage alpha3 (virus) / Genus: Microvirus / Production host: Escherichia coli (E. coli) / References: UniProt: P08767
#3: Protein Major Spike Protein G / G protein / GPG / Coordinate model: Cα atoms only


Mass: 19598.990 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage alpha3 (virus) / Genus: Microvirus / Production host: Escherichia coli (E. coli) / References: UniProt: P31281
#4: Protein Scaffolding Protein B / / GPB / Coordinate model: Cα atoms only


Mass: 8150.042 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage alpha3 (virus) / Genus: Microvirus / Production host: Escherichia coli (E. coli)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Procapsid of the bacteriophage alpha3 / Type: VIRUS
Details of virusDetails: Assembly intermediate called the Procapsid / Host category: BACTERIA(EUBACTERIA) / Isolate: SPECIES
Natural hostOrganism: Escherichia coli / Strain: slyD
Buffer solutionName: 10 mM Tris and 1 mM EDTA / pH: 7.5 / Details: 10 mM Tris and 1 mM EDTA
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Crystal grow
*PLUS
Method: other / Details: cryo-electron microscopy

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Electron microscopy imaging

MicroscopyModel: FEI/PHILIPS CM200FEG/ST / Date: Oct 16, 1998
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 38000 X / Calibrated magnification: 40000 X / Nominal defocus max: 1600 nm / Nominal defocus min: 3200 nm / Cs: 2 mm
Specimen holderTemperature: 88 K / Tilt angle max: 0 ° / Tilt angle min: 0 °
Image recordingElectron dose: 0.017 e/Å2 / Film or detector model: KODAK SO-163 FILM

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Processing

EM software
IDNameCategoryDetails
1EMfitmodel fitting
2RobEMparticle selectionPurdue Program
3EM3DR3D reconstruction
CTF correctionDetails: CTF correction was done for each particle.
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: Model based polar fourier transform (Projection matching).
Resolution: 16 Å / Num. of particles: 2378 / Nominal pixel size: 1.84 Å / Actual pixel size: 1.75 Å
Magnification calibration: Magnification correction was determined using the F pentamer as found in the mature virion of alpha3 and phix174 and in the closed procapsid as a control. Symmetry related ...Magnification calibration: Magnification correction was determined using the F pentamer as found in the mature virion of alpha3 and phix174 and in the closed procapsid as a control. Symmetry related contacts in the control were found to be 6.3% of all atoms. The pixel size of the reconstruction was adjusted so as to obtain the same number of contacts.
Details: 2378 particles were included in the final reconstruction. The effective resolution is 15.0-16.0A. Higher resolution was not possible because of the tendency of particles to orient themselves ...Details: 2378 particles were included in the final reconstruction. The effective resolution is 15.0-16.0A. Higher resolution was not possible because of the tendency of particles to orient themselves non-randomly. Only CA coordinates are presented in the entry.
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Target criteria: Best fit criterion used by the program EMfit is based on the average value of the density at all atomic sites in the fitted protein, the lack of atoms in negative density, and the ...Target criteria: Best fit criterion used by the program EMfit is based on the average value of the density at all atomic sites in the fitted protein, the lack of atoms in negative density, and the absence of symmetry related atomic clashes.
Details: METHOD--General search followed by a climb procedure REFINEMENT PROTOCOL--rigid molecule fit using the program EMfit
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
11M06

1m06

11M061PDBexperimental model
21CD3

1cd3

11CD32PDBexperimental model
RefinementHighest resolution: 16 Å
Refinement stepCycle: LAST / Highest resolution: 16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1179 0 0 0 1179

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