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Basic information

Entry
Database: PDB / ID: 5m3l
TitleSingle-particle cryo-EM using alignment by classification (ABC): the structure of Lumbricus terrestris hemoglobin
Components
  • (Extracellular globin- ...) x 3
  • (Extracellular hemoglobin linker ...) x 2
  • Hemoglobin chain d1
  • Hemoglobin linker chain L1
KeywordsOXYGEN TRANSPORT / Lumbricus 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
Lipocalin - #620 / Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #1520 / Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #1530 / Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #1540 / 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 ...Lipocalin - #620 / Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #1520 / Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #1530 / Single alpha-helices involved in coiled-coils or other helix-helix interfaces - #1540 / 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 / Myoglobin-like, M family globin domain / 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. / Low-density lipoprotein receptor domain class A / Low-density lipoprotein (LDL) receptor class A repeat / LDL receptor-like superfamily / Single alpha-helices involved in coiled-coils or other helix-helix interfaces / Globin/Protoglobin / Globins / Globin-like / Globin / Globin / Globin domain profile. / Helix non-globular / Lipocalin / Globin-like superfamily / Special / Beta Barrel / Orthogonal Bundle / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsAfanasyev, P. / Linnemayr-Seer, C. / Ravelli, R.B.G. / Matadeen, R. / De Carlo, S. / Alewijnse, B. / Portugal, R.V. / Pannu, N.S. / Schatz, M. / van Heel, M.
Funding support Netherlands, United Kingdom, Brazil, 6items
OrganizationGrant numberCountry
The Dutch ministry of economic affairsCyttron II FES-0908; Netherlands
NanoNextNL of the Government of the Netherlands and 130 partnersHTS&M Initiative: FES-0901; Netherlands
Biotechnology and Biological Sciences Research CouncilBB/G015236/1 United Kingdom
The Netherlands Organization for Scientific ResearchNWO grant: 016.072.321 Netherlands
Brazilian science foundationCNPq-152746/2012-9 Brazil
Brazilian science foundationCNPq-400796/2012-0 Brazil
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
DepositionOct 15, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 13, 2017Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2017Group: Database references / Category: citation / citation_author
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed ..._citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.name
Revision 1.2Oct 17, 2018Group: Data collection / Other / Refinement description / Category: cell / refine
Item: _cell.Z_PDB / _cell.length_a ..._cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c
Revision 1.3Jan 23, 2019Group: Advisory / Data collection ...Advisory / Data collection / Derived calculations / Structure summary
Category: database_PDB_rev / database_PDB_rev_record ...database_PDB_rev / database_PDB_rev_record / entity / pdbx_data_processing_status / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _entity.pdbx_mutation
Revision 1.4Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.5May 8, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

Movie
  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Extracellular globin-4
B: Extracellular globin-2
C: Extracellular globin-3
D: Hemoglobin chain d1
E: Extracellular globin-4
F: Extracellular globin-2
G: Extracellular globin-3
H: Hemoglobin chain d1
I: Extracellular globin-4
J: Extracellular globin-2
K: Extracellular globin-3
L: Hemoglobin chain d1
M: Hemoglobin linker chain L1
N: Extracellular hemoglobin linker L2 subunit
O: Extracellular hemoglobin linker L3 subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)283,37127
Polymers275,97415
Non-polymers7,39812
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area54440 Å2
ΔGint-519 kcal/mol
Surface area110100 Å2
MethodPISA
SymmetryPoint symmetry: (Schoenflies symbol: D6 (2x6 fold dihedral))
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21E
12A
22I
13B
23F
14B
24J
15C
25G
16C
26K
17D
27H
18D
28L
19E
29I
110F
210J
111G
211K
112H
212L

NCS domain segments:

Component-ID: _ / Refine code: _

Dom-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
11ASPASPPROPROAA5 - 1515 - 151
21ASPASPPROPROEE5 - 1515 - 151
12ASPASPPROPROAA5 - 1515 - 151
22ASPASPPROPROII5 - 1515 - 151
13LYSLYSHISHISBB1 - 1451 - 145
23LYSLYSHISHISFF1 - 1451 - 145
14LYSLYSHISHISBB2 - 1442 - 144
24LYSLYSHISHISJJ2 - 1442 - 144
15HISHISLEULEUCC3 - 1513 - 151
25HISHISLEULEUGG3 - 1513 - 151
16HISHISLEULEUCC3 - 1513 - 151
26HISHISLEULEUKK3 - 1513 - 151
17GLUGLULYSLYSDD8 - 1471 - 140
27GLUGLULYSLYSHH8 - 1471 - 140
18GLUGLULYSLYSDD8 - 1471 - 140
28GLUGLULYSLYSLL8 - 1471 - 140
19ASPASPPROPROEE5 - 1515 - 151
29ASPASPPROPROII5 - 1515 - 151
110LYSLYSHISHISFF2 - 1442 - 144
210LYSLYSHISHISJJ2 - 1442 - 144
111HISHISLEULEUGG3 - 1513 - 151
211HISHISLEULEUKK3 - 1513 - 151
112GLUGLULYSLYSHH8 - 1471 - 140
212GLUGLULYSLYSLL8 - 1471 - 140

NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10
11
12

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Components

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Extracellular globin- ... , 3 types, 9 molecules AEIBFJCGK

#1: Protein Extracellular globin-4 / Erythrocruorin / Globin A / Globin IV


Mass: 17566.990 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Lumbricus terrestris (common earthworm) / References: UniProt: P13579
#2: Protein Extracellular globin-2 / Erythrocruorin / Globin AIII / Globin B / Globin II


Mass: 16268.229 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Lumbricus terrestris (common earthworm) / References: UniProt: P02218
#3: Protein Extracellular globin-3 / Erythrocruorin / Extracellular globin III / Globin C


Mass: 17331.793 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Lumbricus terrestris (common earthworm) / References: UniProt: P11069

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Protein , 2 types, 4 molecules DHLM

#4: Protein Hemoglobin chain d1


Mass: 15988.263 Da / Num. of mol.: 3 / Source method: isolated from a natural source / Source: (natural) Lumbricus terrestris (common earthworm) / References: UniProt: O61233
#5: Protein Hemoglobin linker chain L1


Mass: 24936.734 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Lumbricus terrestris (common earthworm) / References: UniProt: Q9GV76

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Extracellular hemoglobin linker ... , 2 types, 2 molecules NO

#6: Protein Extracellular hemoglobin linker L2 subunit


Mass: 25085.037 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Lumbricus terrestris (common earthworm) / References: UniProt: Q2I743
#7: Protein Extracellular hemoglobin linker L3 subunit


Mass: 24486.023 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Lumbricus terrestris (common earthworm) / References: UniProt: Q2I742

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Non-polymers , 1 types, 12 molecules

#8: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C34H32FeN4O4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Giant worm hemoglobin / Type: COMPLEX / Entity ID: all / Source: NATURAL
Molecular weightValue: 3.6 MDa / Experimental value: NO
Source (natural)Organism: Lumbricus terrestris (common earthworm) / Organ: Hemolymph
Buffer solutionpH: 7
Buffer component
IDConc.FormulaBuffer-ID
10.1 MTris-HCl1
21 mMEDTA1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 59000 X / Nominal defocus max: 1200 nm / Nominal defocus min: 1000 nm / Cs: 0.02 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 6 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 5235
Image scansMovie frames/image: 7 / Used frames/image: 2-5

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Processing

SoftwareName: REFMAC / Version: 5.8.0155 / Classification: refinement
EM software
IDNameVersionCategory
1IMAGIC-4Dparticle selection
2EPUimage acquisition
4IMAGIC-4DCTF correction
7UCSF Chimera1.11model fitting
9REFMAC5.8.0155model refinement
10PHENIX1.10.1model refinement
11Coot0.8.6model refinement
12IMAGIC-4Dinitial Euler assignment
13IMAGIC-4Dfinal Euler assignment
14IMAGIC-4Dclassification
15IMAGIC-4D3D reconstruction
CTF correctionDetails: Unsupervised MSA classification of amplitude spectra throughout the full data set ("Full data set CTF correction")
Type: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 319746
SymmetryPoint symmetry: D6 (2x6 fold dihedral)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 1/2 BIT CUT-OFF / Num. of particles: 75000 / Algorithm: EXACT BACK PROJECTION
Details: FSC as per the original definition (Harauz & van Heel 1986).
Num. of class averages: 75000 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
RefinementResolution: 3.8→146.67 Å / Cor.coef. Fo:Fc: 0.833 / Cor.coef. Fo:Fc free: 0.841 / SU B: 36.387 / SU ML: 0.487 / Cross valid method: THROUGHOUT / ESU R: 1.633 / ESU R Free: 0.598
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.33227 4656 5 %RANDOM
Rwork0.33093 ---
obs0.331 87730 100 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso mean: 129.873 Å2
Baniso -1Baniso -2Baniso -3
1--2.08 Å2-1.18 Å2-0.54 Å2
2---0.22 Å2-3.25 Å2
3---2.3 Å2
Refinement stepCycle: 1 / Total: 19015
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.01919530
ELECTRON MICROSCOPYr_bond_other_d0.0030.0218262
ELECTRON MICROSCOPYr_angle_refined_deg1.4911.97426641
ELECTRON MICROSCOPYr_angle_other_deg1.023341589
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.09452379
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.79322.733849
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.167153044
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.07615142
ELECTRON MICROSCOPYr_chiral_restr0.0990.22900
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.0222295
ELECTRON MICROSCOPYr_gen_planes_other0.0040.024877
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it8.47112.7659561
ELECTRON MICROSCOPYr_mcbond_other8.47212.7649560
ELECTRON MICROSCOPYr_mcangle_it13.93819.14411925
ELECTRON MICROSCOPYr_mcangle_other13.93719.14511926
ELECTRON MICROSCOPYr_scbond_it9.48213.7859969
ELECTRON MICROSCOPYr_scbond_other9.48213.7859970
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other15.71220.20814716
ELECTRON MICROSCOPYr_long_range_B_refined23.06423355
ELECTRON MICROSCOPYr_long_range_B_other23.06423356
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A83620.08
12E83620.08
21A83660.06
22I83660.06
31B79080.07
32F79080.07
41B78960.07
42J78960.07
51C82700.07
52G82700.07
61C82640.08
62K82640.08
71D79380.08
72H79380.08
81D79940.07
82L79940.07
91E83680.08
92I83680.08
101F78860.05
102J78860.05
111G84200.07
112K84200.07
121H80400.08
122L80400.08
LS refinement shellResolution: 3.8→3.899 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.539 355 -
Rwork0.55 6516 -
obs--100 %

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