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Basic information

Entry
Database: PDB / ID: 5tc1
TitleIn situ structures of the genome and genome-delivery apparatus in ssRNA bacteriophage MS2
Components
  • Capsid protein
  • Maturation protein
  • phage MS2 genome
Keywordsviral protein/rna / asymmetric cryoEM reconstruction / ssRNA genome structure / genome-delivery apparatus / genome-capsid interactions / viral protein-rna complex
Function / homology
Function and homology information


viral genome circularization / virion attachment to host cell pilus / negative regulation of viral translation / T=3 icosahedral viral capsid / virion component / regulation of translation / structural molecule activity / RNA binding / identical protein binding
Similarity search - Function
Assembly protein / Phage maturation protein / MS2 Viral Coat Protein / MS2 Viral Coat Protein / Levivirus coat protein / Levivirus coat protein / Bacteriophage RNA-type, capsid / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / RNA (> 1000) / Maturation protein A / Capsid protein
Similarity search - Component
Biological speciesEnterobacteria phage MS2 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDai, X.H. / Li, Z.H. / Lai, M. / Shu, S. / Du, Y.S. / Zhou, Z.H. / Sun, R.
CitationJournal: Nature / Year: 2017
Title: In situ structures of the genome and genome-delivery apparatus in a single-stranded RNA virus.
Authors: Xinghong Dai / Zhihai Li / Mason Lai / Sara Shu / Yushen Du / Z Hong Zhou / Ren Sun /
Abstract: Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump ...Packaging of the genome into a protein capsid and its subsequent delivery into a host cell are two fundamental processes in the life cycle of a virus. Unlike double-stranded DNA viruses, which pump their genome into a preformed capsid, single-stranded RNA (ssRNA) viruses, such as bacteriophage MS2, co-assemble their capsid with the genome; however, the structural basis of this co-assembly is poorly understood. MS2 infects Escherichia coli via the host 'sex pilus' (F-pilus); it was the first fully sequenced organism and is a model system for studies of translational gene regulation, RNA-protein interactions, and RNA virus assembly. Its positive-sense ssRNA genome of 3,569 bases is enclosed in a capsid with one maturation protein monomer and 89 coat protein dimers arranged in a T = 3 icosahedral lattice. The maturation protein is responsible for attaching the virus to an F-pilus and delivering the viral genome into the host during infection, but how the genome is organized and delivered is not known. Here we describe the MS2 structure at 3.6 Å resolution, determined by electron-counting cryo-electron microscopy (cryoEM) and asymmetric reconstruction. We traced approximately 80% of the backbone of the viral genome, built atomic models for 16 RNA stem-loops, and identified three conserved motifs of RNA-coat protein interactions among 15 of these stem-loops with diverse sequences. The stem-loop at the 3' end of the genome interacts extensively with the maturation protein, which, with just a six-helix bundle and a six-stranded β-sheet, forms a genome-delivery apparatus and joins 89 coat protein dimers to form a capsid. This atomic description of genome-capsid interactions in a spherical ssRNA virus provides insight into genome delivery via the host sex pilus and mechanisms underlying ssRNA-capsid co-assembly, and inspires speculation about the links between nucleoprotein complexes and the origins of viruses.
History
DepositionSep 13, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 7, 2016Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2017Group: Database references
Revision 1.2Jan 18, 2017Group: Database references
Revision 1.3Jan 25, 2017Group: Source and taxonomy
Revision 1.4Nov 8, 2017Group: Data processing / Derived calculations / Category: em_software / pdbx_struct_assembly
Item: _em_software.name / _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details
Revision 1.5Jul 18, 2018Group: Data collection / Category: em_imaging_optics / em_software
Item: _em_imaging_optics.energyfilter_name / _em_software.name
Revision 1.6Nov 20, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_database_related / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Capsid protein
B: Capsid protein
C: Capsid protein
D: Capsid protein
E: Capsid protein
F: Capsid protein
G: Capsid protein
H: Capsid protein
M: Maturation protein
R: phage MS2 genome


Theoretical massNumber of molelcules
Total (without water)1,302,18910
Polymers1,302,18910
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29040 Å2
ΔGint-181 kcal/mol
Surface area117910 Å2
DetailsThe MS2 virion contains an ssRNA chain of the genome enclosed by a capsid built from 178 copies of coat protein and a monomer of the maturation protein. Our structure shows that the maturation protein breaks the icosahedral symmetry of the capsid and imparts structural changes to some of its neighboring coat proteins (chains D, E, F, G, H in this model). All other 173 copies of the coat protein conform to the icosahedral symmetry and have same structure with the crystallographic structure of MS2 capsid with PDB ID 2MS2. Only three of these 173 chains are included in the model (chains A, B, C) for reference.

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Components

#1: Protein
Capsid protein / Coat protein


Mass: 13869.659 Da / Num. of mol.: 8 / Source method: isolated from a natural source
Details: The capsid of MS2 contains 178 copies of the coat protein arranged as 89 dimers in a T=3 icosahedral lattice. Structure of the capsid has been solved by crystallography with icosahedral ...Details: The capsid of MS2 contains 178 copies of the coat protein arranged as 89 dimers in a T=3 icosahedral lattice. Structure of the capsid has been solved by crystallography with icosahedral symmetry applied (PDB ID 2MS2, also included as chains A, B, C in this model). Chains D, E, F, G, H are coat proteins that have slightly different structures in the asymmetric cryoEM reconstruction compared to the crystallographic structure. Structures of the other 170 copies of the coat protein are the same with 2MS2, and thus are not included due to the limited number of chain IDs.
Source: (natural) Enterobacteria phage MS2 (virus) / References: UniProt: P03612
#2: Protein Maturation protein / MP / Assembly protein / A protein


Mass: 44030.934 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Details: The capsid of MS2 contains a single copy of the maturation protein. Our structure shows that it replaces a coat protein dimer at one of the 2-fold icosahedral symmetry axes. Function of the ...Details: The capsid of MS2 contains a single copy of the maturation protein. Our structure shows that it replaces a coat protein dimer at one of the 2-fold icosahedral symmetry axes. Function of the maturation protein is to attach the MS2 virion to the host (E. coli) F-pili and deliver the ssRNA viral genome into the host during infection. of the icosahedral capsid. 178 copies of the coat protein arranged as 89 dimers in a T=3 icosahedral lattice. Structure of the capsid has been solved by crystallography with icosahedral symmetry applied (PDB ID 2MS2, also included as chains A, B, C in this model). Chains D, E, F, G, H are coat proteins that have slightly different structures in the asymmetric cryoEM reconstruction compared to the crystallographic structure. Structures of the other 170 copies of the coat protein are the same with 2MS2, and thus are not included due to the limited number of chain IDs.
Source: (natural) Enterobacteria phage MS2 (virus) / References: UniProt: P03610
#3: RNA chain phage MS2 genome


Mass: 1147200.750 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Enterobacteria phage MS2 (virus) / References: GenBank: 15081
Has protein modificationY

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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

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Enterobacteria phage MS2VIRUSThe viral stock was obtained from ATCC (ATCC number 15597-B1) and cultured in Escherichia coli strain C-3000 (ATCC 15597).all0NATURAL
2capsid shell of Enterobacteria Phage MS2COMPLEXThe capsid of MS2 is composed of 178 copies of the coat protein and one single copy of the maturation protein.#1-#21NATURAL
3coat protein of Enterobacteria Phage MS2COMPLEXThe 178 copies of the coat protein are organized as 89 dimers in a T=3 icosahedral lattice.#12NATURAL
4maturation protein of Enterobacteria Phage MS2COMPLEXThe single copy of maturation protein in the capsid shell of MS2 is located at one of the 2-fold symmetry axes and it replaces a coat protein dimer at this position.#22NATURAL
5the ssRNA genome of Enterobacteria Phage MS2COMPLEXThe genome of MS2 is a single-stranded RNA with 3569 bases. Our asymmetric cryoEM reconstruction of the MS2 virion shows that its ssRNA genome is well organized and has multiple contacts with the capsid shell via tens of RNA stem-loop structures.#31NATURAL
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
113.6 MDaNO
222.5 MDaNO
330.014 MDaNO
440.044 MDaNO
551.1 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Enterobacteria phage MS2 (virus)329852
32Enterobacteria phage MS2 (virus)329852
43Enterobacteria phage MS2 (virus)329852
54Enterobacteria phage MS2 (virus)329852
65Enterobacteria phage MS2 (virus)329852
Details of virus
IDEntity assembly-IDEmptyEnvelopedIsolateType
11NONOSPECIESVIRION
22
33
44
55
Natural host
IDEntity assembly-IDOrganismNcbi tax-IDStrain
11Escherichia coli562C-3000
12
13
14
15
Virus shell
IDEntity assembly-IDNameDiameter (nm)Triangulation number (T number)
11capsid2703
12
13
14
15
Buffer solutionpH: 7.4 / Details: pH7.4 PBS
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: EFTEM mode with Gatan GIF energy filter.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Calibrated magnification: 47170 X / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 79 K
Image recordingAverage exposure time: 5.8 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 6080
EM imaging opticsEnergyfilter name: GIF / Energyfilter upper: 20 eV / Energyfilter lower: 20 eV
Image scansSampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 29 / Used frames/image: 1-14

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Processing

EM software
IDNameVersionCategoryDetails
2Leginonimage acquisition
4CTFFIND3CTF correction
7UCSF Chimera1.1model fitting
9FREALIGN9.11initial Euler assignment
10FREALIGN9.11final Euler assignment
11RELION1.4classification
12FREALIGN9.113D reconstruction
13PHENIXdev_1827model refinementreal space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 360000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 339718 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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