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- EMDB-21012: A small terminase protein from a thermophilic phage with a fixed ... -

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Basic information

Entry
Database: EMDB / ID: EMD-21012
TitleA small terminase protein from a thermophilic phage with a fixed helix-turn-helix geometry, symmetric
Map dataCryo-EM reconstruction of the thermophilic bacteriophage P74-26 small terminase- symmetric
Sample
  • Complex: Gene product 83 of the Thermus thermophilus bacteriophage P74-26
    • Protein or peptide: Small terminase protein
Keywordssmall terminase / bacteriophage / helix-turn-helix / motor / VIRAL PROTEIN
Function / homology: / Phage G20C small terminase, N-terminal domain / Phage G20C small terminase C-terminal domain / Phage G20C small terminase N-terminal domain-containing protein
Function and homology information
Biological speciesThermus virus P74-26
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsHayes JA / Hilbert BJ
Funding support United States, 2 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)1817338 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1F31GM121019-01A1 United States
CitationJournal: J Biol Chem / Year: 2020
Title: A thermophilic phage uses a small terminase protein with a fixed helix-turn-helix geometry.
Authors: Janelle A Hayes / Brendan J Hilbert / Christl Gaubitz / Nicholas P Stone / Brian A Kelch /
Abstract: Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular ...Tailed bacteriophages use a DNA-packaging motor to encapsulate their genome during viral particle assembly. The small terminase (TerS) component of this DNA-packaging machinery acts as a molecular matchmaker that recognizes both the viral genome and the main motor component, the large terminase (TerL). However, how TerS binds DNA and the TerL protein remains unclear. Here we identified gp83 of the thermophilic bacteriophage P74-26 as the TerS protein. We found that TerS oligomerizes into a nonamer that binds DNA, stimulates TerL ATPase activity, and inhibits TerL nuclease activity. A cryo-EM structure of TerS revealed that it forms a ring with a wide central pore and radially arrayed helix-turn-helix domains. The structure further showed that these helix-turn-helix domains, which are thought to bind DNA by wrapping the double helix around the ring, are rigidly held in an orientation distinct from that seen in other TerS proteins. This rigid arrangement of the putative DNA-binding domain imposed strong constraints on how TerS can bind DNA. Finally, the TerS structure lacked the conserved C-terminal β-barrel domain used by other TerS proteins for binding TerL. This suggests that a well-ordered C-terminal β-barrel domain is not required for TerS to carry out its matchmaking function. Our work highlights a thermophilic system for studying the role of small terminase proteins in viral maturation and presents the structure of TerS, revealing key differences between this thermophilic phage and its mesophilic counterparts.
History
DepositionNov 20, 2019-
Header (metadata) releaseFeb 12, 2020-
Map releaseFeb 12, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v1i
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21012.png

Downloads & links

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Map

FileDownload / File: emd_21012.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the thermophilic bacteriophage P74-26 small terminase- symmetric
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 256 pix.
= 271.104 Å		1.06 Å/pix.
x 256 pix.
= 271.104 Å		1.06 Å/pix.
x 256 pix.
= 271.104 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.059 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.040228855 - 0.14541368
Average (Standard dev.)0.00052043545 (±0.004927456)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.104 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0591.0591.059
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.104271.104271.104
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0400.1450.001

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Supplemental data

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Half map: Half map 1 of the 9-fold symmetric cryoEM...

Fileemd_21012_half_map_1.map
AnnotationHalf map 1 of the 9-fold symmetric cryoEM reconstruction of the small terminase protein from the thermophilic bacteriophage P74-26.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of the 9-fold symmetric cryoEM...

Fileemd_21012_half_map_2.map
AnnotationHalf map 2 of the 9-fold symmetric cryoEM reconstruction of the small terminase protein from the thermophilic bacteriophage P74-26.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Gene product 83 of the Thermus thermophilus bacteriophage P74-26

EntireName: Gene product 83 of the Thermus thermophilus bacteriophage P74-26
Components
  • Complex: Gene product 83 of the Thermus thermophilus bacteriophage P74-26
    • Protein or peptide: Small terminase protein

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Supramolecule #1: Gene product 83 of the Thermus thermophilus bacteriophage P74-26

SupramoleculeName: Gene product 83 of the Thermus thermophilus bacteriophage P74-26
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermus virus P74-26
Molecular weightTheoretical: 171 KDa

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Macromolecule #1: Small terminase protein

MacromoleculeName: Small terminase protein / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Thermus virus P74-26
Molecular weightTheoretical: 19.080887 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString:
GPHMSVSFRD RVLKLYLLGF DPSEIAQTLS LDAKRKVTEE EVLHVLAEAR ELLSALPSLE DIRAEVGQAL ERARIFQKDL LAIYQNMLR NYNAMMEGLT EHPDGTPVIG VRPADIAAMA DRIMKIDQER ITALLNSLKV LGHVGSTTAG ALPSATELVS V EELVAEVA DETPKT

UniProtKB: Phage G20C small terminase N-terminal domain-containing protein

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.4 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chloride
20.0 mMTris-HClTris(hydroxymethyl)aminomethane hydrochloride
0.015 %A8-35A8-35
GridPretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER / Details: 20 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse (PDI= 1.000)

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 2822 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.6 µm / Calibrated defocus min: 1.4000000000000001 µm / Calibrated magnification: 13000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ab initio, cisTEM
Final reconstructionApplied symmetry - Point group: C9 (9 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Software - details: Beta / Number images used: 84860
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 3.0) / Software - details: Beta
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Software - details: Beta
Final 3D classificationNumber classes: 6 / Avg.num./class: 85000 / Software - Name: RELION (ver. 3.0) / Software - details: Beta
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4xvn


Chain - Chain ID: A / Chain - Residue range: 1-137 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6v1i:
Cryo-EM reconstruction of the thermophilic bacteriophage P74-26 small terminase- symmetric

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