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- EMDB-3434: Single-particle cryo-EM using alignment by classification (ABC):L... -

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Basic information

Entry
Database: EMDB / ID: EMD-3434
TitleSingle-particle cryo-EM using alignment by classification (ABC):Lumbricus terrestris hemoglobin at near-atomic resolution
Map dataWorm hemoglobin cryo-EM 3D reconstruction sharpened and auto-masked
Sample
  • Sample: Hemoglobin purified from Lumbricus Terrestris
  • Protein or peptide: Hemoglobin
KeywordsLumbricus terrestris / hemoglobin / oxygen carrier / erythrocruorin
Function / homology
Function and homology information


hemoglobin complex / oxygen carrier activity / oxygen binding / iron ion binding / heme binding / extracellular region / metal ion binding
Similarity search - Function
Annelid erythrocruorin linker subunit, C-terminal / Erythrocruorin linker subunit, C-terminal superfamily / Extracellular hemoglobin linker subunit, heterodimerisation domain / Annelid erythrocruorin linker subunit C-terminus / Globin, extracellular / Erythrocruorin / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. / LDL-receptor class A (LDLRA) domain profile. ...Annelid erythrocruorin linker subunit, C-terminal / Erythrocruorin linker subunit, C-terminal superfamily / Extracellular hemoglobin linker subunit, heterodimerisation domain / Annelid erythrocruorin linker subunit C-terminus / Globin, extracellular / Erythrocruorin / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A, conserved site / LDL-receptor class A (LDLRA) domain signature. / LDL-receptor class A (LDLRA) domain profile. / Myoglobin-like, M family globin domain / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A repeat / LDL receptor-like superfamily / Globin/Protoglobin / Globin family profile. / Globin / Globin / Globin-like superfamily
Similarity search - Domain/homology
Extracellular globin / Extracellular globin-2 / Extracellular globin-3 / Extracellular globin-4 / Extracellular hemoglobin linker L3 subunit / Extracellular hemoglobin linker L2 subunit / Hemoglobin linker chain L1
Similarity search - Component
Biological speciesLumbricus terrestris (common earthworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsAfanasyev P / Linnemayr-Seer C / Ravelli RBG / Matadeen R / De Carlo S / Alewijnse B / Portugal RV / Pannu NS / Schatz M / van Heel M
CitationJournal: IUCrJ / Year: 2017
Title: Single-particle cryo-EM using alignment by classification (ABC): the structure of haemoglobin.
Authors: Pavel Afanasyev / Charlotte Seer-Linnemayr / Raimond B G Ravelli / Rishi Matadeen / Sacha De Carlo / Bart Alewijnse / Rodrigo V Portugal / Navraj S Pannu / Michael Schatz / Marin van Heel /
Abstract: Single-particle cryogenic electron microscopy (cryo-EM) can now yield near-atomic resolution structures of biological complexes. However, the reference-based alignment algorithms commonly used in ...Single-particle cryogenic electron microscopy (cryo-EM) can now yield near-atomic resolution structures of biological complexes. However, the reference-based alignment algorithms commonly used in cryo-EM suffer from reference bias, limiting their applicability (also known as the 'Einstein from random noise' problem). Low-dose cryo-EM therefore requires robust and objective approaches to reveal the structural information contained in the extremely noisy data, especially when dealing with small structures. A reference-free pipeline is presented for obtaining near-atomic resolution three-dimensional reconstructions from heterogeneous ('four-dimensional') cryo-EM data sets. The methodologies integrated in this pipeline include camera correction, movie-based full-data-set contrast transfer function determination, movie-alignment algorithms, (Fourier-space) multivariate statistical data compression and unsupervised classification, 'random-startup' three-dimensional reconstructions, four-dimensional structural refinements and Fourier shell correlation criteria for evaluating anisotropic resolution. The procedures exclusively use information emerging from the data set itself, without external 'starting models'. Euler-angle assignments are performed by angular reconstitution rather than by the inherently slower projection-matching approaches. The comprehensive 'ABC-4D' pipeline is based on the two-dimensional reference-free 'alignment by classification' (ABC) approach, where similar images in similar orientations are grouped by unsupervised classification. Some fundamental differences between X-ray crystallography single-particle cryo-EM data collection and data processing are discussed. The structure of the giant haemoglobin from at a global resolution of ∼3.8 Å is presented as an example of the use of the ABC-4D procedure.
History
DepositionMay 11, 2016-
Header (metadata) releaseJun 15, 2016-
Map releaseJul 26, 2017-
UpdateJul 26, 2017-
Current statusJul 26, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5m3l
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5m3l
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3434.tif

Downloads & links

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Map

FileDownload / File: emd_3434.map.gz / Format: CCP4 / Size: 173.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationWorm hemoglobin cryo-EM 3D reconstruction sharpened and auto-masked
Voxel sizeX=Y=Z: 1.11 Å
Density
Contour LevelBy AUTHOR: 1.6 / Movie #1: 1.6
Minimum - Maximum-6.93016148 - 10.0
Average (Standard dev.)-0.00004977 (±0.51959878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-180-180-180
Dimensions360360360
Spacing360360360
CellA=B=C: 399.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.111.111.11
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z399.600399.600399.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-180-180-180
NC/NR/NS360360360
D min/max/mean-6.93010.000-0.000

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Supplemental data

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Segmentation: Automatic mask applied to final 3D reconstruction

AnnotationAutomatic mask applied to final 3D reconstruction
Fileemd_3434_msk_1.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 : Hemoglobin purified from Lumbricus Terrestris

EntireName: Hemoglobin purified from Lumbricus Terrestris
Components
  • Sample: Hemoglobin purified from Lumbricus Terrestris
  • Protein or peptide: Hemoglobin

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Supramolecule #1000: Hemoglobin purified from Lumbricus Terrestris

SupramoleculeName: Hemoglobin purified from Lumbricus Terrestris / type: sample / ID: 1000 / Oligomeric state: Dodecamer / Number unique components: 1
Molecular weightTheoretical: 3.6 MDa

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Macromolecule #1: Hemoglobin

MacromoleculeName: Hemoglobin / type: protein_or_peptide / ID: 1 / Name.synonym: Erythrocruorin / Number of copies: 12 / Oligomeric state: Dodecamer / Recombinant expression: No
Source (natural)Organism: Lumbricus terrestris (common earthworm) / synonym: Common Earthworm / Tissue: hemolymph
Molecular weightTheoretical: 3.6 MDa

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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

BufferpH: 7 / Details: 0.1 M Tris-HCl buffer, 1 mM EDTA
GridDetails: Quantifoil grid (R2/2, Quantifoil Micro Tools GmbH)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: 2.5 s blot time

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.02 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateMay 25, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 5235 / Average electron dose: 40 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Phase flipping of micrograph patches (ctf2d-find)
Final two d classificationNumber classes: 85000
Final angle assignmentDetails: Imagic D6 asymmetric triangle: 0Final reconstructionApplied symmetry - Point group: D6 (2x6 fold dihedral) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: OTHER / Software - Name: Imagic-4D / Number images used: 85000
DetailsReference-free Alignment By Classification (ABC-4D). Camera correction, CTF determination, particle-picking, MSA unsupervised classification, 3D reconstruction, all in IMAGIC-4D.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2gtl

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5m3l:
Single-particle cryo-EM using alignment by classification (ABC): the structure of Lumbricus terrestris hemoglobin

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