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- EMDB-10912: Cryo-EM structure of T7 bacteriophage DNA translocation gp15-gp16... -

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Basic information

Entry
Database: EMDB / ID: EMD-10912
TitleCryo-EM structure of T7 bacteriophage DNA translocation gp15-gp16 core complex intermediate assembly
Map datat7 tail complex
Sample
  • Complex: gp15-gp16 core complex
    • Protein or peptide: Internal virion protein gp15
    • Protein or peptide: Peptidoglycan transglycosylase gp16
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: 3.0 Å
AuthorsPerez-Ruiz M / Pulido-Cid M / Luque-Ortega JR / Cuervo A / Carrascosa JL
Funding support Spain, 3 items
OrganizationGrant numberCountry
Spanish Ministry of Science, Innovation, and UniversitiesBFU 2014-54181 Spain
Spanish Ministry of Science, Innovation, and UniversitiesSEV-2013-0347 Spain
Spanish Ministry of Science, Innovation, and UniversitiesBES-2015-073615 Spain
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Assisted assembly of bacteriophage T7 core components for genome translocation across the bacterial envelope.
Authors: Mar Pérez-Ruiz / Mar Pulido-Cid / Juan Román Luque-Ortega / José María Valpuesta / Ana Cuervo / José L Carrascosa /
Abstract: In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the family, in which the tail is not long enough to traverse the bacterial ...In most bacteriophages, genome transport across bacterial envelopes is carried out by the tail machinery. In viruses of the family, in which the tail is not long enough to traverse the bacterial wall, it has been postulated that viral core proteins assembled inside the viral head are translocated and reassembled into a tube within the periplasm that extends the tail channel. Bacteriophage T7 infects , and despite extensive studies, the precise mechanism by which its genome is translocated remains unknown. Using cryo-electron microscopy, we have resolved the structure of two different assemblies of the T7 DNA translocation complex composed of the core proteins gp15 and gp16. Gp15 alone forms a partially folded hexamer, which is further assembled upon interaction with gp16 into a tubular structure, forming a channel that could allow DNA passage. The structure of the gp15-gp16 complex also shows the location within gp16 of a canonical transglycosylase motif involved in the degradation of the bacterial peptidoglycan layer. This complex docks well in the tail extension structure found in the periplasm of T7-infected bacteria and matches the sixfold symmetry of the phage tail. In such cases, gp15 and gp16 that are initially present in the T7 capsid eightfold-symmetric core would change their oligomeric state upon reassembly in the periplasm. Altogether, these results allow us to propose a model for the assembly of the core translocation complex in the periplasm, which furthers understanding of the molecular mechanism involved in the release of T7 viral DNA into the bacterial cytoplasm.
History
DepositionApr 23, 2020-
Header (metadata) releaseSep 8, 2021-
Map releaseSep 8, 2021-
UpdateSep 8, 2021-
Current statusSep 8, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.006
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6yt5
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10912.png

Downloads & links

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Map

FileDownload / File: emd_10912.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationt7 tail complex
Voxel sizeX=Y=Z: 1.047 Å
Density
Contour LevelBy AUTHOR: 0.006 / Movie #1: 0.006
Minimum - Maximum-0.011678981 - 0.15056488
Average (Standard dev.)9.248733e-05 (±0.0035224585)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 335.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0471.0471.047
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z335.040335.040335.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0120.1510.000

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Supplemental data

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Sample components

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Entire : gp15-gp16 core complex

EntireName: gp15-gp16 core complex
Components
  • Complex: gp15-gp16 core complex
    • Protein or peptide: Internal virion protein gp15
    • Protein or peptide: Peptidoglycan transglycosylase gp16

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Supramolecule #1: gp15-gp16 core complex

SupramoleculeName: gp15-gp16 core complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia phage T7 (virus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: C41
Molecular weightExperimental: 1.4 MDa

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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: 88.377219 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDPSSMSKIE SALQAAQPGL SRLRGGAGGM GYRAATTQAE QPRSSLLDTI GRFAKAGAD MYTAKEQRAR DLADERSNEI IRKLTPEQRR EALNNGTLLY QDDPYAMEAL RVKTGRNAAY LVDDDVMQKI K EGVFRTRE ...String:
MRGSHHHHHH GMASMTGGQQ MGRDLYDDDD KDPSSMSKIE SALQAAQPGL SRLRGGAGGM GYRAATTQAE QPRSSLLDTI GRFAKAGAD MYTAKEQRAR DLADERSNEI IRKLTPEQRR EALNNGTLLY QDDPYAMEAL RVKTGRNAAY LVDDDVMQKI K EGVFRTRE EMEEYRHSRL QEGAKVYAEQ FGIDPEDVDY QRGFNGDITE RNISLYGAHD NFLSQQAQKG AIMNSRVELN GV LQDPDML RRPDSADFFE KYIDNGLVTG AIPSDAQATQ LISQAFSDAS SRAGGADFLM RVGDKKVTLN GATTTYRELI GEE QWNALM VTAQRSQFET DAKLNEQYRL KINSALNQED PRTAWEMLQG IKAELDKVQP DEQMTPQREW LISAQEQVQN QMNA WTKAQ AKALDDSMKS MNKLDVIDKQ FQKRINGEWV STDFKDMPVN ENTGEFKHSD MVNYANKKLA EIDSMDIPDG AKDAM KLKY LQADSKDGAF RTAIGTMVTD AGQEWSAAVI NGKLPERTPA MDALRRIRNA DPQLIAALYP DQAELFLTMD MMDKQG IDP QVILDADRLT VKRSKEQRFE DDKAFESALN ASKAPEIARM PASLRESARK IYDSVKYRSG NESMAMEQMT KFLKEST YT FTGDDVDGDT VGVIPKNMMQ VNSDPKSWEQ GRDILEEARK GIIASNPWIT NKQLTMYSQG DSIYLMDTTG QVRVRYDK E LLSKVWSENQ KKLEEKAREK ALADVNKRAP IVAATKAREA AAKRVREKRK QTPKFIYGRK E

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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: 146.693094 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGHHHHHHHH HHSSGHIEGR HMMDKYDKNV PSDYDGLFQK AADANGVSYD LLRKVAWTES RFVPTAKSKT GPLGMMQFTK ATAKALGLR VTDGPDDDRL NPELAINAAA KQLAGLVGKF DGDELKAALA YNQGEGRLGN PQLEAYSKGD FASISEEGRN Y MRNLLDVA ...String:
MGHHHHHHHH HHSSGHIEGR HMMDKYDKNV PSDYDGLFQK AADANGVSYD LLRKVAWTES RFVPTAKSKT GPLGMMQFTK ATAKALGLR VTDGPDDDRL NPELAINAAA KQLAGLVGKF DGDELKAALA YNQGEGRLGN PQLEAYSKGD FASISEEGRN Y MRNLLDVA KSPMAGQLET FGGITPKGKG IPAEVGLAGI GHKQKVTQEL PESTSFDVKG IEQEATAKPF AKDFWETHGE TL DEYNSRS TFFGFKNAAE AELSNSVAGM AFRAGRLDNG FDVFKDTITP TRWNSHIWTP EELEKIRTEV KNPAYINVVT GGS PENLDD LIKLANENFE NDSRAAEAGL GAKLSAGIIG AGVDPLSYVP MVGVTGKGFK LINKALVVGA ESAALNVASE GLRT SVAGG DADYAGAALG GFVFGAGMSA ISDAVAAGLK RSKPEAEFDN EFIGPMMRLE ARETARNANS ADLSRMNTEN MKFEG EHNG VPYEDLPTER GAVVLHDGSV LSASNPINPK TLKEFSEVDP EKAARGIKLA GFTEIGLKTL GSDDADIRRV AIDLVR SPT GMQSGASGKF GATASDIHER LHGTDQRTYN DLYKAMSDAM KDPEFSTGGA KMSREETRYT IYRRAALAIE RPELQKA LT PSERIVMDII KRHFDTKREL MENPAIFGNT KAVSIFPESR HKGTYVPHVY DRHAKALMIQ RYGAEGLQEG IARSWMNS Y VSRPEVKARV DEMLKELHGV KEVTPEMVEK YAMDKAYGIS HSDQFTNSSI IEENIEGLVG IENNSFLEAR NLFDSDLSI TMPDGQQFSV NDLRDFDMFR IMPAYDRRVN GDIAIMGSTG KTTKELKDEI LALKAKAEGD GKKTGEVHAL MDTVKILTGR ARRNQDTVW ETSLRAINDL GFFAKNAYMG AQNITEIAGM IVTGNVRALG HGIPILRDTL YKSKPVSAKE LKELHASLFG K EVDQLIRP KRADIVQRLR EATDTGPAVA NIVGTLKYST QELAARSPWT KLLNGTTNYL LDAARQGMLG DVISATLTGK TT RWEKEGF LRGASVTPEQ MAGIKSLIKE HMVRGEDGKF TVKDKQAFSM DPRAMDLWRL ADKVADEAML RPHKVSLQDS HAF GALGKM VMQFKSFTIK SLNSKFLRTF YDGYKNNRAI DAALSIITSM GLAGGFYAMA AHVKAYALPK EKRKEYLERA LDPT MIAHA ALSRSSQLGA PLAMVDLVGG VLGFESSKMA RSTILPKDTV KERDPNKPYT SREVMGAMGS NLLEQMPSAG FVANV GATL MNAAGVVNSP NKATEQDFMT GLMNSTKELV PNDPLTQQLV LKIYEANGVN LRERRK

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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

Concentration1.5 mg/mL
BufferpH: 8
Component:
ConcentrationFormula
100.0 mMTris
100.0 mMNaCitrate
10.0 mMMgCl2
GridModel: Quantifoil R2/2 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 47619 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 2 / Number real images: 5506 / Average exposure time: 8.0 sec. / Average electron dose: 40.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware: (Name: Gctf, RELION)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 72882
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

1sly

chain_id: A

4cfo

chain_id: A
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6yt5:
Cryo-EM structure of T7 bacteriophage DNA translocation gp15-gp16 core complex intermediate assembly

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