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- EMDB-22680: Structure of T7 DNA ejectosome periplasmic tunnel -

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Basic information

Entry
Database: EMDB / ID: EMD-22680
TitleStructure of T7 DNA ejectosome periplasmic tunnel
Map dataPost-process map from Relion-3.0
Sample
  • Complex: Multisubunit T7 DNA ejectosome periplasmic tunnel with 6x copies of Gp15 and 6x copies of Gp16
    • Protein or peptide: Internal virion protein gp15
    • Protein or peptide: Peptidoglycan transglycosylase gp16
  • Ligand: water
KeywordsT7 / ejectosome / ejection protein / genome-delivery / podoviridae / VIRAL PROTEIN
Function / homology
Function and homology information


symbiont genome ejection through host cell envelope / host cell periplasmic space / : / symbiont entry into host cell via disruption of host cell wall peptidoglycan / lytic transglycosylase activity / symbiont genome ejection through host cell envelope, short tail mechanism / symbiont entry into host cell via disruption of host cell envelope / peptidoglycan metabolic process / symbiont entry into host / virion component ...symbiont genome ejection through host cell envelope / host cell periplasmic space / : / symbiont entry into host cell via disruption of host cell wall peptidoglycan / lytic transglycosylase activity / symbiont genome ejection through host cell envelope, short tail mechanism / symbiont entry into host cell via disruption of host cell envelope / peptidoglycan metabolic process / symbiont entry into host / virion component / killing of cells of another organism / hydrolase activity / defense response to bacterium / host cell plasma membrane / membrane
Similarity search - Function
Internal virion protein Gp16 / Internal virion protein Gp15 / Prokaryotic transglycosylase, active site / Prokaryotic transglycosylases signature. / Transglycosylase SLT domain 1 / Transglycosylase SLT domain / Lysozyme-like domain superfamily
Similarity search - Domain/homology
Internal virion protein gp15 / Peptidoglycan transglycosylase gp16
Similarity search - Component
Biological speciesEscherichia phage T7 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsSwanson N / Cingolani G
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM100888 United States
CitationJournal: Mol Cell / Year: 2021
Title: Cryo-EM structure of the periplasmic tunnel of T7 DNA-ejectosome at 2.7 Å resolution.
Authors: Nicholas A Swanson / Ravi K Lokareddy / Fenglin Li / Chun-Feng David Hou / Sebastian Leptihn / Mikhail Pavlenok / Michael Niederweis / Ruth A Pumroy / Vera Y Moiseenkova-Bell / Gino Cingolani /
Abstract: Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome ...Hershey and Chase used bacteriophage T2 genome delivery inside Escherichia coli to demonstrate that DNA, not protein, is the genetic material. Seventy years later, our understanding of viral genome delivery in prokaryotes remains limited, especially for short-tailed phages of the Podoviridae family. These viruses expel mysterious ejection proteins found inside the capsid to form a DNA-ejectosome for genome delivery into bacteria. Here, we reconstitute the phage T7 DNA-ejectosome components gp14, gp15, and gp16 and solve the periplasmic tunnel structure at 2.7 Å resolution. We find that gp14 forms an outer membrane pore, gp15 assembles into a 210 Å hexameric DNA tube spanning the host periplasm, and gp16 extends into the host cytoplasm forming a ∼4,200 residue hub. Gp16 promotes gp15 oligomerization, coordinating peptidoglycan hydrolysis, DNA binding, and lipid insertion. The reconstituted gp15:gp16 complex lacks channel-forming activity, suggesting that the pore for DNA passage forms only transiently during genome ejection.
History
DepositionSep 16, 2020-
Header (metadata) releaseJul 28, 2021-
Map releaseJul 28, 2021-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k5c
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22680.png

Downloads & links

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Map

FileDownload / File: emd_22680.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-process map from Relion-3.0
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 352 pix.
= 380.16 Å		1.08 Å/pix.
x 352 pix.
= 380.16 Å		1.08 Å/pix.
x 352 pix.
= 380.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.0267 / Movie #1: 0.025
Minimum - Maximum-0.17808002 - 0.2799669
Average (Standard dev.)-0.0000007178213 (±0.0076619512)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 380.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z352352352
origin x/y/z0.0000.0000.000
length x/y/z380.160380.160380.160
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS352352352
D min/max/mean-0.1780.280-0.000

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Supplemental data

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Sample components

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Entire : Multisubunit T7 DNA ejectosome periplasmic tunnel with 6x copies ...

EntireName: Multisubunit T7 DNA ejectosome periplasmic tunnel with 6x copies of Gp15 and 6x copies of Gp16
Components
  • Complex: Multisubunit T7 DNA ejectosome periplasmic tunnel with 6x copies of Gp15 and 6x copies of Gp16
    • Protein or peptide: Internal virion protein gp15
    • Protein or peptide: Peptidoglycan transglycosylase gp16
  • Ligand: water

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Supramolecule #1: Multisubunit T7 DNA ejectosome periplasmic tunnel with 6x copies ...

SupramoleculeName: Multisubunit T7 DNA ejectosome periplasmic tunnel with 6x copies of Gp15 and 6x copies of Gp16
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Escherichia phage T7 (virus)
Molecular weightTheoretical: 590 KDa

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Macromolecule #1: Internal virion protein gp15

MacromoleculeName: Internal virion protein gp15 / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia phage T7 (virus)
Molecular weightTheoretical: 84.454008 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSKIESALQA AQPGLSRLRG GAGGMGYRAA TTQAEQPRSS LLDTIGRFAK AGADMYTAKE QRARDLADER SNEIIRKLTP EQRREALNN GTLLYQDDPY AMEALRVKTG RNAAYLVDDD VMQKIKEGVF RTREEMEEYR HSRLQEGAKV YAEQFGIDPE D VDYQRGFN ...String:
MSKIESALQA AQPGLSRLRG GAGGMGYRAA TTQAEQPRSS LLDTIGRFAK AGADMYTAKE QRARDLADER SNEIIRKLTP EQRREALNN GTLLYQDDPY AMEALRVKTG RNAAYLVDDD VMQKIKEGVF RTREEMEEYR HSRLQEGAKV YAEQFGIDPE D VDYQRGFN GDITERNISL YGAHDNFLSQ QAQKGAIMNS RVELNGVLQD PDMLRRPDSA DFFEKYIDNG LVTGAIPSDA QA TQLISQA FSDASSRAGG ADFLMRVGDK KVTLNGATTT YRELIGEEQW NALMVTAQRS QFETDAKLNE QYRLKINSAL NQE DPRTAW EMLQGIKAEL DKVQPDEQMT PQREWLISAQ EQVQNQMNAW TKAQAKALDD SMKSMNKLDV IDKQFQKRIN GEWV STDFK DMPVNENTGE FKHSDMVNYA NKKLAEIDSM DIPDGAKDAM KLKYLQADSK DGAFRTAIGT MVTDAGQEWS AAVIN GKLP ERTPAMDALR RIRNADPQLI AALYPDQAEL FLTMDMMDKQ GIDPQVILDA DRLTVKRSKE QRFEDDKAFE SALNAS KAP EIARMPASLR ESARKIYDSV KYRSGNESMA MEQMTKFLKE STYTFTGDDV DGDTVGVIPK NMMQVNSDPK SWEQGRD IL EEARKGIIAS NPWITNKQLT MYSQGDSIYL MDTTGQVRVR YDKELLSKVW SENQKKLEEK AREKALADVN KRAPIVAA T KAREAAAKRV REKRKQTPKF IYGRKE

UniProtKB: Internal virion protein gp15

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Macromolecule #2: Peptidoglycan transglycosylase gp16

MacromoleculeName: Peptidoglycan transglycosylase gp16 / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
EC number: Lyases; Carbon-oxygen lyases; Acting on polysaccharides
Source (natural)Organism: Escherichia phage T7 (virus)
Molecular weightTheoretical: 144.028219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDKYDKNVPS DYDGLFQKAA DANGVSYDLL RKVAWTESRF VPTAKSKTGP LGMMQFTKAT AKALGLRVTD GPDDDRLNPE LAINAAAKQ LAGLVGKFDG DELKAALAYN QGEGRLGNPQ LEAYSKGDFA SISEEGRNYM RNLLDVAKSP MAGQLETFGG I TPKGKGIP ...String:
MDKYDKNVPS DYDGLFQKAA DANGVSYDLL RKVAWTESRF VPTAKSKTGP LGMMQFTKAT AKALGLRVTD GPDDDRLNPE LAINAAAKQ LAGLVGKFDG DELKAALAYN QGEGRLGNPQ LEAYSKGDFA SISEEGRNYM RNLLDVAKSP MAGQLETFGG I TPKGKGIP AEVGLAGIGH KQKVTQELPE STSFDVKGIE QEATAKPFAK DFWETHGETL DEYNSRSTFF GFKNAAEAEL SN SVAGMAF RAGRLDNGFD VFKDTITPTR WNSHIWTPEE LEKIRTEVKN PAYINVVTGG SPENLDDLIK LANENFENDS RAA EAGLGA KLSAGIIGAG VDPLSYVPMV GVTGKGFKLI NKALVVGAES AALNVASEGL RTSVAGGDAD YAGAALGGFV FGAG MSAIS DAVAAGLKRS KPEAEFDNEF IGPMMRLEAR ETARNANSAD LSRMNTENMK FEGEHNGVPY EDLPTERGAV VLHDG SVLS ASNPINPKTL KEFSEVDPEK AARGIKLAGF TEIGLKTLGS DDADIRRVAI DLVRSPTGMQ SGASGKFGAT ASDIHE RLH GTDQRTYNDL YKAMSDAMKD PEFSTGGAKM SREETRYTIY RRAALAIERP ELQKALTPSE RIVMDIIKRH FDTKREL ME NPAIFGNTKA VSIFPESRHK GTYVPHVYDR HAKALMIQRY GAEGLQEGIA RSWMNSYVSR PEVKARVDEM LKELHGVK E VTPEMVEKYA MDKAYGISHS DQFTNSSIIE ENIEGLVGIE NNSFLEARNL FDSDLSITMP DGQQFSVNDL RDFDMFRIM PAYDRRVNGD IAIMGSTGKT TKELKDEILA LKAKAEGDGK KTGEVHALMD TVKILTGRAR RNQDTVWETS LRAINDLGFF AKNAYMGAQ NITEIAGMIV TGNVRALGHG IPILRDTLYK SKPVSAKELK ELHASLFGKE VDQLIRPKRA DIVQRLREAT D TGPAVANI VGTLKYSTQE LAARSPWTKL LNGTTNYLLD AARQGMLGDV ISATLTGKTT RWEKEGFLRG ASVTPEQMAG IK SLIKEHM VRGEDGKFTV KDKQAFSMDP RAMDLWRLAD KVADEAMLRP HKVSLQDSHA FGALGKMVMQ FKSFTIKSLN SKF LRTFYD GYKNNRAIDA ALSIITSMGL AGGFYAMAAH VKAYALPKEK RKEYLERALD PTMIAHAALS RSSQLGAPLA MVDL VGGVL GFESSKMARS TILPKDTVKE RDPNKPYTSR EVMGAMGSNL LEQMPSAGFV ANVGATLMNA AGVVNSPNKA TEQDF MTGL MNSTKELVPN DPLTQQLVLK IYEANGVNLR ERRK

UniProtKB: Peptidoglycan transglycosylase gp16

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 219 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration2.2 mg/mL
BufferpH: 8.5
Component:
ConcentrationFormulaName
100.0 mMTris/HClTris buffer
10.0 mMMgCl2magnesium chloride
100.0 mMNa3C6H5O7sodium citrate

Details: Solutions were made fresh and filtered.
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 120 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 6 seconds before plunging.
DetailsThis sample was monodisperse, but high concentration. Some preferred orientation.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8601 / Average exposure time: 3.4 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4700000
Details: Manually picked 2000 particles using Relion GUI, followed by 2D-Classification to select templates, then Relion auto-picking was used.
Startup modelType of model: NONE
Details: Generated ab initio with Relion-3.0-b from this dataset
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0-b) / Number images used: 208024
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-b) / Details: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0-b) / Details: RELION
Final 3D classificationNumber classes: 6 / Avg.num./class: 75000 / Software - Name: RELION (ver. 3.0-b)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 45.1 / Target criteria: 0.92
Output model

PDB-7k5c:
Structure of T7 DNA ejectosome periplasmic tunnel

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