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- EMDB-10476: Neck of native GTA particle computed with C12 symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-10476
TitleNeck of native GTA particle computed with C12 symmetry
Map dataneck of native GTA particle computed with C12 symmetry
Sample
  • Complex: Rhodobacter capsulatus DE442 gene transfer agent connector
    • Protein or peptide: Adaptor protein Rcc01688Adapter
    • Protein or peptide: Phage portal protein, HK97 family
Function / homologyPhage conserved hypothetical protein / Phage portal protein, HK97 / Bacteriophage/Gene transfer agent portal protein / Phage portal protein / Gene transfer agent protein / Putative gene transfer agent portal protein
Function and homology information
Biological speciesRhodobacter capsulatus DE442 (bacteria) / Rhodobacter capsulatus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsBardy P / Fuzik T / Hrebik D / Pantucek R / Beatty JT / Plevka P
Funding support Czech Republic, 7 items
OrganizationGrant numberCountry
Ministry of Education (Czech Republic)LQ1601 Czech Republic
Ministry of Education (Czech Republic)LM2011033 Czech Republic
European Regional Development FundCZ.1.05/1.1.00/02.0070 Czech Republic
Czech Science Foundation18-17810S Czech Republic
Czech Science Foundation15-21631Y Czech Republic
Grant Agency of the Czech Republic18-13064S Czech Republic
European Molecular Biology Organization3041 Czech Republic
CitationJournal: Nat Commun / Year: 2020
Title: Structure and mechanism of DNA delivery of a gene transfer agent.
Authors: Pavol Bárdy / Tibor Füzik / Dominik Hrebík / Roman Pantůček / J Thomas Beatty / Pavel Plevka /
Abstract: Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the ...Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.
History
DepositionNov 11, 2019-
Header (metadata) releaseJul 22, 2020-
Map releaseJul 22, 2020-
UpdateJul 22, 2020-
Current statusJul 22, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.039
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.039
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6te8
  • Surface level: 0.039
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6te8
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_10476.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationneck of native GTA particle computed with C12 symmetry
Voxel sizeX=Y=Z: 1.063 Å
Density
Contour LevelBy AUTHOR: 0.039 / Movie #1: 0.039
Minimum - Maximum-0.10756149 - 0.187955
Average (Standard dev.)0.0005116354 (±0.0068911114)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 318.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0631.0631.063
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z318.900318.900318.900
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1080.1880.001

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Supplemental data

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Sample components

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Entire : Rhodobacter capsulatus DE442 gene transfer agent connector

EntireName: Rhodobacter capsulatus DE442 gene transfer agent connector
Components
  • Complex: Rhodobacter capsulatus DE442 gene transfer agent connector
    • Protein or peptide: Adaptor protein Rcc01688Adapter
    • Protein or peptide: Phage portal protein, HK97 family

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Supramolecule #1: Rhodobacter capsulatus DE442 gene transfer agent connector

SupramoleculeName: Rhodobacter capsulatus DE442 gene transfer agent connector
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: special 5-fold vertex of the capsid, interconnecting capsid to tail
Source (natural)Organism: Rhodobacter capsulatus DE442 (bacteria)
Molecular weightTheoretical: 760 KDa

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Macromolecule #1: Adaptor protein Rcc01688

MacromoleculeName: Adaptor protein Rcc01688 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rhodobacter capsulatus (bacteria)
Molecular weightTheoretical: 20.956354 KDa
SequenceString: MMLNEVTAVP GTALPVAEFR DHLRLGTGFA DLGAEDAALL SYLRAAIAAI EGRTAKALIS RGFRLALTAW RWGDMQTLPI APVATVTAL RLVDAAGVET PVAAGWRLVP DMARPRIEAL GAMLPMIPTG GRVEIDFTAG FGASWSALPV DLAQAVFLLA A QYYELRHD ...String:
MMLNEVTAVP GTALPVAEFR DHLRLGTGFA DLGAEDAALL SYLRAAIAAI EGRTAKALIS RGFRLALTAW RWGDMQTLPI APVATVTAL RLVDAAGVET PVAAGWRLVP DMARPRIEAL GAMLPMIPTG GRVEIDFTAG FGASWSALPV DLAQAVFLLA A QYYELRHD GAAEGGAMPF GVMALIERWR TVRVLGGRP

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Macromolecule #2: Phage portal protein, HK97 family

MacromoleculeName: Phage portal protein, HK97 family / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rhodobacter capsulatus (bacteria)
Molecular weightTheoretical: 42.589668 KDa
SequenceString: MGLNFFRKAA PEVRTEPVAE RKASVTGRIV AMASGAGRPV WGPRDTVSLM RTGFAGNPVG FRSVKLIAEA TAAVPLICQD AERRYEIHP VLDLLRRPNA GQGRAELFEA LIGQILLSGN GYLEAVCPEP GVPRELHVLR SDRMAVVPGA DGWPVGYDYT V GGRKHRFD ...String:
MGLNFFRKAA PEVRTEPVAE RKASVTGRIV AMASGAGRPV WGPRDTVSLM RTGFAGNPVG FRSVKLIAEA TAAVPLICQD AERRYEIHP VLDLLRRPNA GQGRAELFEA LIGQILLSGN GYLEAVCPEP GVPRELHVLR SDRMAVVPGA DGWPVGYDYT V GGRKHRFD MTGHPDPICH IKSFHPTDDH YGLSPMQAAA VALDVHNAAS AWSKALLDNA ARPSGAIIYK GADGQGVLAP EQ YERLIFE METHHQGARN AGRPMLLEGG LDWKPMGFSP SDMEFHETKA AAAREIALAF GVPPMLIGIP GDATYANYAE ANR AFYRLT VLPLLTRVSA ALAWWLSGYL GAQIELKPDL DQVPALAVER DQLWARIGAA GFLSNSEKRV LLGLPPT

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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

Concentration20 mg/mL
BufferpH: 7.8
Component:
ConcentrationNameFormula
10.0 mMTris
1.0 mMNaClSodium chloride
1.0 mMMgCl2
1.0 mMCaCl2
0.01 mg/mlBovine serum albumin

Details: G-buffer, doi: 10.1016/0003-9861(77)90508-2
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 11.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / 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 opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: -3.0 µm / Nominal defocus min: -1.0 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 3114 / Average exposure time: 1.0 sec. / Average electron dose: 42.75 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 41516
CTF correctionSoftware - Name: RELION (ver. 2.1)
Startup modelType of model: INSILICO MODEL
In silico model: stochastic gradient descent in RELION 2.1 de novo
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 2.1) / Details: one class discarded
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 41151
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6te8:
Neck of native GTA particle computed with C12 symmetry

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