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- EMDB-24002: Cryo-EM map of the FLP cargo within the T. maritima encapsulin -

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Basic information

Entry
Database: EMDB / ID: EMD-24002
TitleCryo-EM map of the FLP cargo within the T. maritima encapsulin
Map dataSymmetry expansion and local classification of one FLP cargo protein within the T. maritima encapsulin
Sample
  • Organelle or cellular component: Cryo-EM map of FLP cargo protein within the T. maritima encapsulin
Function / homologyType 1 encapsulin shell protein / Encapsulating protein for peroxidase / encapsulin nanocompartment / Hydrolases; Acting on peptide bonds (peptidases) / peptidase activity / iron ion transport / intracellular iron ion homeostasis / proteolysis / Type 1 encapsulin shell protein
Function and homology information
Biological speciesThermotoga maritima (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsLaFrance BJ / Savage DF / Nogales E
Funding support United States, 2 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States)DE-SC00016240 United States
National Science Foundation (NSF, United States)GRFP-1106400 United States
CitationJournal: Sci Rep / Year: 2021
Title: The encapsulin from Thermotoga maritima is a flavoprotein with a symmetry matched ferritin-like cargo protein.
Authors: Benjamin J LaFrance / Caleb Cassidy-Amstutz / Robert J Nichols / Luke M Oltrogge / Eva Nogales / David F Savage /
Abstract: Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based 'organelles' found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ...Bacterial nanocompartments, also known as encapsulins, are an emerging class of protein-based 'organelles' found in bacteria and archaea. Encapsulins are virus-like icosahedral particles comprising a ~ 25-50 nm shell surrounding a specific cargo enzyme. Compartmentalization is thought to create a unique chemical environment to facilitate catalysis and isolate toxic intermediates. Many questions regarding nanocompartment structure-function remain unanswered, including how shell symmetry dictates cargo loading and to what extent the shell facilitates enzymatic activity. Here, we explore these questions using the model Thermotoga maritima nanocompartment known to encapsulate a redox-active ferritin-like protein. Biochemical analysis revealed the encapsulin shell to possess a flavin binding site located at the interface between capsomere subunits, suggesting the shell may play a direct and active role in the function of the encapsulated cargo. Furthermore, we used cryo-EM to show that cargo proteins use a form of symmetry-matching to facilitate encapsulation and define stoichiometry. In the case of the Thermotoga maritima encapsulin, the decameric cargo protein with fivefold symmetry preferentially binds to the pentameric-axis of the icosahedral shell. Taken together, these observations suggest the shell is not simply a passive barrier-it also plays a significant role in the structure and function of the cargo enzyme.
History
DepositionMay 14, 2021-
Header (metadata) releaseDec 8, 2021-
Map releaseDec 8, 2021-
UpdateDec 8, 2021-
Current statusDec 8, 2021Processing 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
  • Download
  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_24002.png

Downloads & links

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Map

FileDownload / File: emd_24002.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSymmetry expansion and local classification of one FLP cargo protein within the T. maritima encapsulin
Voxel sizeX=Y=Z: 1.64 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.025
Minimum - Maximum-0.08251401 - 0.18174626
Average (Standard dev.)0.0005741794 (±0.008908013)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 419.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.641.641.64
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z419.840419.840419.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0830.1820.001

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Supplemental data

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Additional map: C1 reconstruction of T. maritima encapsulin showing all...

Fileemd_24002_additional_1.map
AnnotationC1 reconstruction of T. maritima encapsulin showing all FLP cargo densities present in the shell
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM map of FLP cargo protein within the T. maritima encapsulin

EntireName: Cryo-EM map of FLP cargo protein within the T. maritima encapsulin
Components
  • Organelle or cellular component: Cryo-EM map of FLP cargo protein within the T. maritima encapsulin

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Supramolecule #1: Cryo-EM map of FLP cargo protein within the T. maritima encapsulin

SupramoleculeName: Cryo-EM map of FLP cargo protein within the T. maritima encapsulin
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Thermotoga maritima (bacteria)
Molecular weightTheoretical: 1.83 MDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)

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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

Concentration2.0 mg/mL
BufferpH: 7.4
Details: 20 mM Tris-HCl, pH 7.4, 150 mM NaCl and 5 mM 2-mercaptoethanol
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot force.

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 1 / Number real images: 1276 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.8000000000000003 µm / Nominal defocus min: 1.5 µm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN

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Image processing

DetailsImages were saved in counting mode using leginon software, and subsequently processed in RELION.
Particle selectionNumber selected: 86124
Details: 1000 particles were manually selected from random micrographs. 2D classification was performed on these 1000 particles, and 2D classes were used for relion auto picking
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Details: CTFFind4 implemented within RELION/3.0, CTFRefine was also performed after initial reconstruction was achieved.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3dkt


Details: Original T. maritima crystal structure from Sutter et al 'molmap' function in Chimera was used to generate surface model. Initial model was lowpass filtered to 30A for initial ...Details: Original T. maritima crystal structure from Sutter et al 'molmap' function in Chimera was used to generate surface model. Initial model was lowpass filtered to 30A for initial reconstruction, and once high-resolution was achieved, a 5A model was used to assure the correct handedness.
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: It should be noted that this number of particles is not 'true' single particles, but the result of symmetry expansion. Thus, some of these particles are unique orientations of the same ...Details: It should be noted that this number of particles is not 'true' single particles, but the result of symmetry expansion. Thus, some of these particles are unique orientations of the same encapsulin. Also, the resolution of 4A is for the entire shell with cargo, however, the cargo is estimated to be 7-8A.
Number images used: 249296
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationSoftware - Name: RELION (ver. 3.1)
Details: The icosahedral particles were symmetry expanded and a localized alignment free classification was performed at one 5-fold vertex to better classify a single FLP cargo protein. 3 iterations ...Details: The icosahedral particles were symmetry expanded and a localized alignment free classification was performed at one 5-fold vertex to better classify a single FLP cargo protein. 3 iterations of this were performed resulting in the final reconstruction deposited herein.

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Atomic model buiding 1

Initial modelPDB ID:

3dkt


Chain - Chain ID: A
DetailsThe homology model for the encapsulated FLP was created using Biozentrum's SWISS-MODEL, with 5DA5 as the reference structure. This was then rigid body docked into the FLP map using ChimeraX followed by PHENIX. The final model can be found in the supplementary materials of this manuscript at 'FLP-pentamer_model.py'
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 110

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