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- EMDB-21024: Single-particle cryo-EM reconstruction of mouse heavy chain apofe... -

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Basic information

Entry
Database: EMDB / ID: EMD-21024
TitleSingle-particle cryo-EM reconstruction of mouse heavy chain apoferritin at 200 keV
Map dataSingle-particle cryo-EM reconstruction of mouse heavy chain apoferritin using 200 keV
Sample
  • Complex: Heavy chain apoferritin from mouse
    • Protein or peptide: Ferritin heavy chain
  • Ligand: water
Keywordshomo-24-mer / storage / globular / METAL BINDING PROTEIN / OXIDOREDUCTASE
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / autolysosome / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / autolysosome / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation / ferric iron binding / ferrous iron binding / iron ion transport / immune response / iron ion binding / negative regulation of cell population proliferation / mitochondrion / extracellular region / identical protein binding / cytoplasm
Similarity search - Function
Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily
Similarity search - Domain/homology
Ferritin heavy chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.75 Å
AuthorsWu M / Lander GC
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)DP2EB020402 United States
CitationJournal: J Struct Biol X / Year: 2020
Title: Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV.
Authors: Mengyu Wu / Gabriel C Lander / Mark A Herzik /
Abstract: Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination ...Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as ∼1.7 Å resolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 kV electrons for high-resolution single-particle cryo-EM and applications such as structure-based drug design.
History
DepositionNov 21, 2019-
Header (metadata) releaseFeb 12, 2020-
Map releaseFeb 12, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / 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 radius
  • Surface level: 0.039
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v21
  • Surface level: 0.039
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21024.png

Downloads & links

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Map

FileDownload / File: emd_21024.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle cryo-EM reconstruction of mouse heavy chain apoferritin using 200 keV
Voxel sizeX=Y=Z: 0.562 Å
Density
Contour LevelBy AUTHOR: 0.039 / Movie #1: 0.039
Minimum - Maximum-0.14008248 - 0.2509239
Average (Standard dev.)0.0000059833096 (±0.012311106)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 215.80798 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5620.5620.562
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z215.808215.808215.808
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/NS384384384
D min/max/mean-0.1400.2510.000

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Supplemental data

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Mask #1

Fileemd_21024_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_21024_additional.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Even half map

Fileemd_21024_half_map_1.map
AnnotationEven half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Odd half map

Fileemd_21024_half_map_2.map
AnnotationOdd half 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 : Heavy chain apoferritin from mouse

EntireName: Heavy chain apoferritin from mouse
Components
  • Complex: Heavy chain apoferritin from mouse
    • Protein or peptide: Ferritin heavy chain
  • Ligand: water

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Supramolecule #1: Heavy chain apoferritin from mouse

SupramoleculeName: Heavy chain apoferritin from mouse / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Recombinantly expressed and purified from E. coli BL21(DE3)pLys cells
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 505 KDa

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Macromolecule #1: Ferritin heavy chain

MacromoleculeName: Ferritin heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 24 / Enantiomer: LEVO / EC number: ferroxidase
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 20.304818 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
SPSQVRQNYH QDAEAAINRQ INLELYASYV YLSMSCYFDR DDVALKNFAK YFLHQSHEER EHAEKLMKLQ NQRGGRIFLQ DIKKPDRDD WESGLNAMEC ALHLEKSVNQ SLLELHKLAT DKNDPHLCDF IETYYLSEQV KSIKELGDHV TNLRKMGAPE A GMAEYLFD KHTLGH

UniProtKB: Ferritin heavy chain

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 1783 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

Concentration4 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
30.0 mMHEPES
150.0 mMSodium ChlorideNaClSodium chloride
1.0 mMDTT
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 6 sec. / Pretreatment - Atmosphere: OTHER / Details: 15 Watts
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: 3 uL of sample/grid was manually blotted for 4 seconds prior to immediate plunge-freezing in liquid nitrogen-cooled ethane..

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 73000
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: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Digitization - Frames/image: 1-90 / Number grids imaged: 1 / Number real images: 1759 / Average exposure time: 9.0 sec. / Average electron dose: 58.0 e/Å2
Details: Images were collected using stage position navigation to target exposure.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 405106
Startup modelType of model: EMDB MAP
EMDB ID:

9599


Details: EMD-9599 was low-pass filtered to 8 angstroms and used as an initial model for 3D refinement
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: O (octahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 1.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1)
Details: A refined spherical aberration value of 2.8 mm was used for the final reconstruction
Number images used: 241878
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3wnw


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Overall B value: 39
Output model

PDB-6v21:
Mouse heavy chain apoferritin determined using single-particle cryo-EM at 200 keV

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