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- EMDB-8707: Negative stain reconstruction of Endoplasmic Reticulum HSP40 co-c... -

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Basic information

Entry
Database: EMDB / ID: EMD-8707
TitleNegative stain reconstruction of Endoplasmic Reticulum HSP40 co-chaperone native ERdj3 tetramer.
Map dataNegative stain reconstruction of native ERdj3 tetramer
Sample
  • Complex: Tetrameric complex of native ERdj3 protein complex
Function / homology
Function and homology information


XBP1(S) activates chaperone genes / misfolded protein binding / positive regulation of ATP-dependent activity / protein folding chaperone complex / IRE1-mediated unfolded protein response / protein maturation / unfolded protein binding / protein folding / endoplasmic reticulum lumen / endoplasmic reticulum / membrane
Similarity search - Function
HSP40/DnaJ peptide-binding / Chaperone DnaJ, C-terminal / DnaJ C terminal domain / Nt-dnaJ domain signature. / DnaJ domain, conserved site / DnaJ domain / DnaJ molecular chaperone homology domain / dnaJ domain profile. / Chaperone J-domain superfamily / DnaJ domain
Similarity search - Domain/homology
DnaJ homolog subfamily B member 11
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 16.9 Å
AuthorsChowdhury S / Lander GC / Chen K-C / Qu S / Noxon IC / Schonhoft JD / Plate L / Powers ET / Kelly JW / Wiseman RL
CitationJournal: Proc Natl Acad Sci U S A / Year: 2017
Title: Structures of Qβ virions, virus-like particles, and the Qβ-MurA complex reveal internal coat proteins and the mechanism of host lysis.
Authors: Zhicheng Cui / Karl V Gorzelnik / Jeng-Yih Chang / Carrie Langlais / Joanita Jakana / Ry Young / Junjie Zhang /
Abstract: In single-stranded RNA bacteriophages (ssRNA phages) a single copy of the maturation protein binds the genomic RNA (gRNA) and is required for attachment of the phage to the host pilus. For the ...In single-stranded RNA bacteriophages (ssRNA phages) a single copy of the maturation protein binds the genomic RNA (gRNA) and is required for attachment of the phage to the host pilus. For the canonical Qβ the maturation protein, A, has an additional role as the lysis protein, by its ability to bind and inhibit MurA, which is involved in peptidoglycan biosynthesis. Here, we determined structures of Qβ virions, virus-like particles, and the Qβ-MurA complex using single-particle cryoelectron microscopy, at 4.7-Å, 3.3-Å, and 6.1-Å resolutions, respectively. We identified the outer surface of the β-region in A as the MurA-binding interface. Moreover, the pattern of MurA mutations that block Qβ lysis and the conformational changes of MurA that facilitate A binding were found to be due to the intimate fit between A and the region encompassing the closed catalytic cleft of substrate-liganded MurA. Additionally, by comparing the Qβ virion with Qβ virus-like particles that lack a maturation protein, we observed a structural rearrangement in the capsid coat proteins that is required to package the viral gRNA in its dominant conformation. Unexpectedly, we found a coat protein dimer sequestered in the interior of the virion. This coat protein dimer binds to the gRNA and interacts with the buried α-region of A, suggesting that it is sequestered during the early stage of capsid formation to promote the gRNA condensation required for genome packaging. These internalized coat proteins are the most asymmetrically arranged major capsid proteins yet observed in virus structures.
History
DepositionApr 26, 2017-
Header (metadata) releaseMay 24, 2017-
Map releaseMay 24, 2017-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0179
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0179
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8707.png

Downloads & links

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Map

FileDownload / File: emd_8707.map.gz / Format: CCP4 / Size: 844.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain reconstruction of native ERdj3 tetramer
Voxel sizeX=Y=Z: 4.1 Å
Density
Contour LevelBy AUTHOR: 0.0179 / Movie #1: 0.0179
Minimum - Maximum-0.08225489 - 0.2763727
Average (Standard dev.)0.000801101 (±0.014040019)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin262626
Dimensions606060
Spacing606060
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.14.14.1
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z246.000246.000246.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS262626
NC/NR/NS606060
D min/max/mean-0.0820.2760.001

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Supplemental data

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Sample components

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Entire : Tetrameric complex of native ERdj3 protein complex

EntireName: Tetrameric complex of native ERdj3 protein complex
Components
  • Complex: Tetrameric complex of native ERdj3 protein complex

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Supramolecule #1: Tetrameric complex of native ERdj3 protein complex

SupramoleculeName: Tetrameric complex of native ERdj3 protein complex / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant cell: SHuffle T7 Express
Molecular weightTheoretical: 153 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.025 mg/mL
BufferpH: 7.5 / Details: 50 mM HEPES, 300 mM NaCl, 1% (v/v)Glycerol
StainingType: NEGATIVE / Material: Uranyl Formate
Details: Sample absorbed on carbon surface was stained with 2% (w/v) Uranyl Formate solution, and finally air-dried after blotting off excess stain.
GridModel: 400 mesh Cu-Rh maxtaform grids (Electron Microscopy Sciences) that were coated with a thin layer of amorphous carbon.
Material: COPPER/RHODIUM / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 15mA current
DetailsMono dispersed protein solution

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureMin: 293.0 K / Max: 295.0 K
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 15.6 µm / Number grids imaged: 2 / Number real images: 980 / Average exposure time: 0.79 sec. / Average electron dose: 35.0 e/Å2
Details: Automated image acquisition software Leginon was used for data collection.

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Image processing

Particle selectionNumber selected: 385141
Details: Difference of Gaussian (DOG)-based automated particle picker, implemented in Appion processing package was used for particle selection.
CTF correctionSoftware - Name: CTFFIND4
Details: Ctffind4 was used for determining the CTF of each micrograph. Phases of each micrographs were flipped before particle extraction.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2qld


Details: Four copies of PDB 2QLD were overlaid on 2D classes and low pass filtered to generate an initial 3D model.
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 16.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 17405
DetailsAutomated image acquisition software Leginon was used for data collection.
FSC plot (resolution estimation)

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