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- EMDB-4597: T=4 quasi symmetric bacterial microcompartment particle with one ... -

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Basic information

Entry
Database: EMDB / ID: EMD-4597
TitleT=4 quasi symmetric bacterial microcompartment particle with one missing pentameric EutN unit
Map data
Sample
  • Complex: bacterial microcompartment particles
    • Protein or peptide: cmcD/EutN BMC-P bacterial microcompartment protein
    • Protein or peptide: cmcC/PduA/ccmK BMC-H bacterial microcompartment protein
Biological speciesKlebsiella pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.75 Å
AuthorsKalnins G
CitationJournal: Nat Commun / Year: 2020
Title: Encapsulation mechanisms and structural studies of GRM2 bacterial microcompartment particles.
Authors: Gints Kalnins / Eva-Emilija Cesle / Juris Jansons / Janis Liepins / Anatolij Filimonenko / Kaspars Tars /
Abstract: Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, ...Bacterial microcompartments (BMCs) are prokaryotic organelles consisting of a protein shell and an encapsulated enzymatic core. BMCs are involved in several biochemical processes, such as choline, glycerol and ethanolamine degradation and carbon fixation. Since non-native enzymes can also be encapsulated in BMCs, an improved understanding of BMC shell assembly and encapsulation processes could be useful for synthetic biology applications. Here we report the isolation and recombinant expression of BMC structural genes from the Klebsiella pneumoniae GRM2 locus, the investigation of mechanisms behind encapsulation of the core enzymes, and the characterization of shell particles by cryo-EM. We conclude that the enzymatic core is encapsulated in a hierarchical manner and that the CutC choline lyase may play a secondary role as an adaptor protein. We also present a cryo-EM structure of a pT = 4 quasi-symmetric icosahedral shell particle at 3.3 Å resolution, and demonstrate variability among the minor shell forms.
History
DepositionFeb 8, 2019-
Header (metadata) releaseDec 25, 2019-
Map releaseDec 25, 2019-
UpdateFeb 12, 2020-
Current statusFeb 12, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0227
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0227
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4597.png

Downloads & links

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Map

FileDownload / File: emd_4597.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.23 Å
Density
Contour LevelBy AUTHOR: 0.0234 / Movie #1: 0.0227
Minimum - Maximum-0.024936749 - 0.06889794
Average (Standard dev.)0.0023785545 (±0.010672679)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 393.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z393.600393.600393.600
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/NS320320320
D min/max/mean-0.0250.0690.002

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Supplemental data

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Half map: #1

Fileemd_4597_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_4597_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : bacterial microcompartment particles

EntireName: bacterial microcompartment particles
Components
  • Complex: bacterial microcompartment particles
    • Protein or peptide: cmcD/EutN BMC-P bacterial microcompartment protein
    • Protein or peptide: cmcC/PduA/ccmK BMC-H bacterial microcompartment protein

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Supramolecule #1: bacterial microcompartment particles

SupramoleculeName: bacterial microcompartment particles / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: bacterial microcompartment particle obtained by recombinant expression of pentameric EutN and hexameric cmcC subunits
Source (natural)Organism: Klebsiella pneumoniae (bacteria) / Strain: MSCL535
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET-Duet1
Molecular weightExperimental: 2.55 MDa

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Macromolecule #1: cmcD/EutN BMC-P bacterial microcompartment protein

MacromoleculeName: cmcD/EutN BMC-P bacterial microcompartment protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MILAKVTGHV VATQKCDELR GSNLLLITRL DDKQQPMKDQ TWVAVDNVGA GMHDIVLAEE YFALNKDRYK AMSVVAIVEK VFRD

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Macromolecule #2: cmcC/PduA/ccmK BMC-H bacterial microcompartment protein

MacromoleculeName: cmcC/PduA/ccmK BMC-H bacterial microcompartment protein
type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Klebsiella pneumoniae (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MKEALGLIET KGLVACIEAA DAMCKAANVE LIGYENVGSG LVTAMVKGDV GAVNAAVDSG VEAAKRIGEV VSSRVIARPH NDIEKIAG

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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 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMNaClSodium chloride
20.0 mMTris-HClTris
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4 seconds before plunging.
DetailsSample was purified with gel filtration

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: -3.0 µm / Nominal defocus min: -1.4 µm / Nominal magnification: 120000
Specialist opticsPhase plate: VOLTA PHASE PLATE
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: COUNTING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number real images: 1316 / Average exposure time: 1.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 62533
CTF correctionSoftware - Name: RELION (ver. 3.0)
Startup modelType of model: INSILICO MODEL
In silico model: Made in Relion 3.0 using stochastic Gradient Descent (SGD) algorithm
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 2 / Avg.num./class: 5820 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 11639
FSC plot (resolution estimation)

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