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- EMDB-3404: Asymmetric cryo-EM reconstruction of phage MS2 reveals genome str... -

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Basic information

Entry
Database: EMDB / ID: EMD-3404
TitleAsymmetric cryo-EM reconstruction of phage MS2 reveals genome structure in situ
Map dataDensity of RNA and A-protein of bacteriophage MS2
Sample
  • Sample: Bacteriophage MS2 RNA and A-protein
  • RNA: Enterobacterio phage MS2, complete genome
KeywordsMS2 / RNA genome / structure / cryo-EM
Biological speciesEnterobacterio phage MS2 (virus)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 10.5 Å
AuthorsKoning RI / Gomez-Blanco J / Akopjana I / Vargas J / Kazaks A / Tars K / Carazo JM / Koster AJ
CitationJournal: Nat Commun / Year: 2016
Title: Asymmetric cryo-EM reconstruction of phage MS2 reveals genome structure in situ.
Authors: Roman I Koning / Josue Gomez-Blanco / Inara Akopjana / Javier Vargas / Andris Kazaks / Kaspars Tars / José María Carazo / Abraham J Koster /
Abstract: In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the ...In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation. The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly.
History
DepositionMar 30, 2016-
Header (metadata) releaseApr 6, 2016-
Map releaseSep 21, 2016-
UpdateOct 12, 2016-
Current statusOct 12, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3404.png

Downloads & links

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Map

FileDownload / File: emd_3404.map.gz / Format: CCP4 / Size: 111 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationDensity of RNA and A-protein of bacteriophage MS2
Voxel sizeX=Y=Z: 1.14 Å
Density
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.42515033 - 0.03816911
Average (Standard dev.)-0.03042487 (±0.08298364)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions310310310
Spacing310310310
CellA=B=C: 353.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.141.141.14
M x/y/z310310310
origin x/y/z0.0000.0000.000
length x/y/z353.400353.400353.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS310310310
D min/max/mean-0.4250.038-0.030

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Supplemental data

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Sample components

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Entire : Bacteriophage MS2 RNA and A-protein

EntireName: Bacteriophage MS2 RNA and A-protein
Components
  • Sample: Bacteriophage MS2 RNA and A-protein
  • RNA: Enterobacterio phage MS2, complete genome

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Supramolecule #1000: Bacteriophage MS2 RNA and A-protein

SupramoleculeName: Bacteriophage MS2 RNA and A-protein / type: sample / ID: 1000 / Details: Purified by gel filtration / Oligomeric state: One copy of genomic RNA and one A-protein / Number unique components: 1
Molecular weightTheoretical: 1.28 MDa

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Macromolecule #1: Enterobacterio phage MS2, complete genome

MacromoleculeName: Enterobacterio phage MS2, complete genome / type: rna / ID: 1
Details: This map is a difference map of the MS2 virion (cryo-EM density map by SPA EMD-3403) and the capsid (178 copies of pbd entry 2MS2)
Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Enterobacterio phage MS2 (virus) / synonym: Phage MS2
Molecular weightTheoretical: 1.28 MDa
SequenceString: GGGTGGGACC CCTTTCGGGG TCCTGCTCAA CTTCCTGTCG AGCTAATGCC ATTTTTAATG TCTTTAGCGA GACGCTACCA TGGCTATCGC TGTAGGTAGC CGGAATTCCA TTCCTAGGAG GTTTGACCTG TGCGAGCTTT TAGTACCCTT GATAGGGAGA ACGAGACCTT ...String:
GGGTGGGACC CCTTTCGGGG TCCTGCTCAA CTTCCTGTCG AGCTAATGCC ATTTTTAATG TCTTTAGCGA GACGCTACCA TGGCTATCGC TGTAGGTAGC CGGAATTCCA TTCCTAGGAG GTTTGACCTG TGCGAGCTTT TAGTACCCTT GATAGGGAGA ACGAGACCTT CGTCCCCTCC GTTCGCGTTT ACGCGGACGG TGAGACTGAA GATAACTCAT TCTCTTTAAA ATATCGTTCG AACTGGACTC CCGGTCGTTT TAACTCGACT GGGGCCAAAA CGAAACAGTG GCACTACCCC TCTCCGTATT CACGGGGGGC GTTAAGTGTC ACATCGATAG ATCAAGGTGC CTACAAGCGA AGTGGGTCAT CGTGGGGTCG CCCGTACGAG GAGAAAGCCG GTTTCGGCTT CTCCCTCGAC GCACGCTCCT GCTACAGCCT CTTCCCTGTA AGCCAAAACT TGACTTACAT CGAAGTGCCG CAGAACGTTG CGAACCGGGC GTCGACCGAA GTCCTGCAAA AGGTCACCCA GGGTAATTTT AACCTTGGTG TTGCTTTAGC AGAGGCCAGG TCGACAGCCT CACAACTCGC GACGCAAACC ATTGCGCTCG TGAAGGCGTA CACTGCCGCT CGTCGCGGTA ATTGGCGCCA GGCGCTCCGC TACCTTGCCC TAAACGAAGA TCGAAAGTTT CGATCAAAAC ACGTGGCCGG CAGGTGGTTG GAGTTGCAGT TCGGTTGGTT ACCACTAATG AGTGATATCC AGGGTGCATA TGAGATGCTT ACGAAGGTTC ACCTTCAAGA GTTTCTTCCT ATGAGAGCCG TACGTCAGGT CGGTACTAAC ATCAAGTTAG ATGGCCGTCT GTCGTATCCA GCTGCAAACT TCCAGACAAC GTGCAACATA TCGCGACGTA TCGTGATATG GTTTTACATA AACGATGCAC GTTTGGCATG GTTGTCGTCT CTAGGTATCT TGAACCCACT AGGTATAGTG TGGGAAAAGG TGCCTTTCTC ATTCGTTGTC GACTGGCTCC TACCTGTAGG TAACATGCTC GAGGGCCTTA CGGCCCCCGT GGGATGCTCC TACATGTCAG GAACAGTTAC TGACGTAATA ACGGGTGAGT CCATCATAAG CGTTGACGCT CCCTACGGGT GGACTGTGGA GAGACAGGGC ACTGCTAAGG CCCAAATCTC AGCCATGCAT CGAGGGGTAC AATCCGTATG GCCAACAACT GGCGCGTACG TAAAGTCTCC TTTCTCGATG GTCCATACCT TAGATGCGTT AGCATTAATC AGGCAACGGC TCTCTAGATA GAGCCCTCAA CCGGAGTTTG AAGCATGGCT TCTAACTTTA CTCAGTTCGT TCTCGTCGAC AATGGCGGAA CTGGCGACGT GACTGTCGCC CCAAGCAACT TCGCTAACGG GGTCGCTGAA TGGATCAGCT CTAACTCGCG TTCACAGGCT TACAAAGTAA CCTGTAGCGT TCGTCAGAGC TCTGCGCAGA ATCGCAAATA CACCATCAAA GTCGAGGTGC CTAAAGTGGC AACCCAGACT GTTGGTGGTG TAGAGCTTCC TGTAGCCGCA TGGCGTTCGT ACTTAAATAT GGAACTAACC ATTCCAATTT TCGCTACGAA TTCCGACTGC GAGCTTATTG TTAAGGCAAT GCAAGGTCTC CTAAAAGATG GAAACCCGAT TCCCTCAGCA ATCGCAGCAA ACTCCGGCAT CTACTAATAG ACGCCGGCCA TTCAAACATG AGGATTACCC ATGTCGAAGA CAACAAAGAA GTTCAACTCT TTATGTATTG ATCTTCCTCG CGATCTTTCT CTCGAAATTT ACCAATCAAT TGCTTCTGTC GCTACTGGAA GCGGTGATCC GCACAGTGAC GACTTTACAG CAATTGCTTA CTTAAGGGAC GAATTGCTCA CAAAGCATCC GACCTTAGGT TCTGGTAATG ACGAGGCGAC CCGTCGTACC TTAGCTATCG CTAAGCTACG GGAGGCGAAT GGTGATCGCG GTCAGATAAA TAGAGAAGGT TTCTTACATG ACAAATCCTT GTCATGGGAT CCGGATGTTT TACAAACCAG CATCCGTAGC CTTATTGGCA ACCTCCTCTC TGGCTACCGA TCGTCGTTGT TTGGGCAATG CACGTTCTCC AACGGTGCTC CTATGGGGCA CAAGTTGCAG GATGCAGCGC CTTACAAGAA GTTCGCTGAA CAAGCAACCG TTACCCCCCG CGCTCTGAGA GCGGCTCTAT TGGTCCGAGA CCAATGTGCG CCGTGGATCA GACACGCGGT CCGCTATAAC GAGTCATATG AATTTAGGCT CGTTGTAGGG AACGGAGTGT TTACAGTTCC GAAGAATAAT AAAATAGATC GGGCTGCCTG TAAGGAGCCT GATATGAATA TGTACCTCCA GAAAGGGGTC GGTGCTTTCA TCAGACGCCG GCTCAAATCC GTTGGTATAG ACCTGAATGA TCAATCGATC AACCAGCGTC TGGCTCAGCA GGGCAGCGTA GATGGTTCGC TTGCGACGAT AGACTTATCG TCTGCATCCG ATTCCATCTC CGATCGCCTG GTGTGGAGTT TTCTCCCACC AGAGCTATAT TCATATCTCG ATCGTATCCG CTCACACTAC GGAATCGTAG ATGGCGAGAC GATACGATGG GAACTATTTT CCACAATGGG AAATGGGTTC ACATTTGAGC TAGAGTCCAT GATATTCTGG GCAATAGTCA AAGCGACCCA AATCCATTTT GGTAACGCCG GAACCATAGG CATCTACGGG GACGATATTA TATGTCCCAG TGAGATTGCA CCCCGTGTGC TAGAGGCACT TGCCTACTAC GGTTTTAAAC CGAATCTTCG TAAAACGTTC GTGTCCGGGC TCTTTCGCGA GAGCTGCGGC GCGCACTTTT ACCGTGGTGT CGATGTCAAA CCGTTTTACA TCAAGAAACC TGTTGACAAT CTCTTCGCCC TGATGCTGAT ATTAAATCGG CTACGGGGTT GGGGAGTTGT CGGAGGTATG TCAGATCCAC GCCTCTATAA GGTGTGGGTA CGGCTCTCCT CCCAGGTGCC TTCGATGTTC TTCGGTGGGA CGGACCTCGC TGCCGACTAC TACGTAGTCA GCCCGCCTAC GGCAGTCTCG GTATACACCA AGACTCCGTA CGGGCGGCTG CTCGCGGATA CCCGTACCTC GGGTTTCCGT CTTGCTCGTA TCGCTCGAGA ACGCAAGTTC TTCAGCGAAA AGCACGACAG TGGTCGCTAC ATAGCGTGGT TCCATACTGG AGGTGAAATC ACCGACAGCA TGAAGTCCGC CGGCGTGCGC GTTATACGCA CTTCGGAGTG GCTAACGCCG GTTCCCACAT TCCCTCAGGA GTGTGGGCCA GCGAGCTCTC CTCGGTAGCT GACCGAGGGA CCCCCGTAAA CGGGGTGGGT GTGCTCGAAA GAGCACGGGT GCGAAAGCGG TCCGGCTCCA CCGAAAGGTG GGCGGGCTTC GGCCCAGGGA CCTCCCCCTA AAGAGAGGAC CCGGGATTCT CCCGATTTGG TAACTAGCTG CTTGGCTAGT TACCACCCA

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1 mg/mL
BufferpH: 7.4 / Details: PBS
StainingType: NEGATIVE / Details: Vitrification
GridDetails: Cu 300 Mesh with quantifoil R2/2 support film, glow discharged in air at 0.2 mbar for 1 minute at 30 mA.
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 70 % / Chamber temperature: 100 K / Instrument: LEICA EM GP
Method: Blotted using filter paper for 1 to 2 seconds before blotting

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 61403 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.02 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
TemperatureMin: 77 K / Max: 97 K / Average: 87 K
Alignment procedureLegacy - Astigmatism: Cs corrector / Legacy - Electron beam tilt params: 15
DateMay 19, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 751 / Average electron dose: 35 e/Å2
Details: Images are average of 7 frames recorded in movie mode on direct electron detector
Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Per image
Final two d classificationNumber classes: 5201
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.5 Å / Resolution method: OTHER / Software - Name: Scipion, Xmipp, CTFFIND3, Relion
Details: This map is a difference map of the MS2 virion (cryo-EM density map by SPA EMD-3403) and the capsid (178 copies of pbd entry 2MS2)
Number images used: 18977
DetailsMovies were aligned using Optical Flow, CTFs were estimated using CTFFIND3, and were used to select the best quality micrographs. A total of 22,441 particles were picked automatically using Xmipp. Particles were classified using 2D reference-free Relion approach. Relion was used for 3D refinement using icosahedral symmetry and gold-standard approach.Chimera was used to retract density of capsid.

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