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Entry
Database: PDB / ID: 5m3l
TitleSingle-particle cryo-EM using alignment by classification (ABC): the structure of Lumbricus terrestris hemoglobin
Descriptor(Extracellular globin- ...) x 3
(Hemoglobin ...) x 2
(Extracellular hemoglobin linker ...) x 2
KeywordsOXYGEN TRANSPORT / Lumbricus terrestris / hemoglobin / oxygen carrier / erythrocruorin / oxygen transport
Specimen sourceLumbricus terrestris / invertebrata / Common earthworm
MethodElectron microscopy (3.8 Å resolution / Particle / Single particle)
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.
CitationIUCrJ, 2017, 4, 678-694

IUCrJ, 2017, 4, 678-694 Yorodumi Papers
Single-particle cryo-EM using alignment by classification (ABC): the structure of Lumbricus terrestris haemoglobin.
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

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 15, 2016 / Release: Sep 13, 2017

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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
Polyers275,97415
Non-polymers7,39812
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)54440
ΔGint (kcal/M)-519
Surface area (Å2)110100
MethodPISA

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Components

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

#1: Polypeptide(L)Extracellular globin-4 / Erythrocruorin / Globin A / Globin IV


Mass: 17566.990 Da / Num. of mol.: 3 / Mutation: D78K / Source: (natural) Lumbricus terrestris / References: UniProt: P13579

Cellular component

Molecular function

#2: Polypeptide(L)Extracellular globin-2 / Erythrocruorin / Globin AIII / Globin B / Globin II


Mass: 16268.229 Da / Num. of mol.: 3 / Mutation: E66D / Source: (natural) Lumbricus terrestris / References: UniProt: P02218

Cellular component

Molecular function

#3: Polypeptide(L)Extracellular globin-3 / Erythrocruorin / Extracellular globin III / Globin C


Mass: 17331.793 Da / Num. of mol.: 3 / Mutation: D49E / Source: (natural) Lumbricus terrestris / References: UniProt: P11069

Cellular component

Molecular function

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Hemoglobin ... , 2 types, 4 molecules DHLM

#4: Polypeptide(L)Hemoglobin chain d1


Mass: 15988.263 Da / Num. of mol.: 3 / Source: (natural) Lumbricus terrestris / References: UniProt: O61233
#5: Polypeptide(L)Hemoglobin linker chain L1


Mass: 24936.734 Da / Num. of mol.: 1 / Source: (natural) Lumbricus terrestris / References: UniProt: Q9GV76

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

#6: Polypeptide(L)Extracellular hemoglobin linker L2 subunit


Mass: 25085.037 Da / Num. of mol.: 1 / Source: (natural) Lumbricus terrestris / References: UniProt: Q2I743
#7: Polypeptide(L)Extracellular hemoglobin linker L3 subunit


Mass: 24486.023 Da / Num. of mol.: 1 / Source: (natural) Lumbricus terrestris / 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 / Formula: C34H32FeN4O4

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: Giant worm hemoglobin / Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8 / Source: NATURAL
Molecular weightValue: 3.6 deg. / Units: MEGADALTONS / Experimental value: NO
Source (natural)Organ: Hemolymph / Organism: Lumbricus terrestris
Buffer solutionpH: 7
Buffer component
IDConc.UnitsFormulaBuffer ID
10.1MTris-HCl1
21mMEDTA1
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 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 59000 / 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) / Number 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
IDNameVersionCategoryImage processing IDImaging IDFitting ID
1IMAGIC-4DPARTICLE SELECTION1
2EPUIMAGE ACQUISITION1
4IMAGIC-4DCTF CORRECTION1
7UCSF Chimera1.11MODEL FITTING1
9REFMAC5.8.0155MODEL REFINEMENT1
10PHENIX1.10.1MODEL REFINEMENT1
11Coot0.8.6MODEL REFINEMENT1
12IMAGIC-4DINITIAL EULER ASSIGNMENT1
13IMAGIC-4DFINAL EULER ASSIGNMENT1
14IMAGIC-4DCLASSIFICATION1
15IMAGIC-4DRECONSTRUCTION1
CTF correctionDetails: Unsupervised MSA classification of amplitude spectra throughout the full data set ("Full data set CTF correction")
Type: PHASE FLIPPING ONLY
Particle selectionNumber of particles selected: 319746
SymmetryPoint symmetry: D6
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 1/2 BIT CUT-OFF / Number of particles: 75000 / Algorithm: EXACT BACK PROJECTION
Details: FSC as per the original definition (Harauz & van Heel 1986).
Number of class averages: 75000 / Symmetry type: POINT
Atomic model buildingRef protocol: RIGID BODY FIT / Ref space: REAL / Target criteria: Cross-correlation coefficient
Refine
Refine IDB iso meanAniso B11Aniso B12Aniso B13Aniso B22Aniso B23Aniso B33Correlation coeff Fo to FcCorrelation coeff Fo to Fc freeDetailsR factor R freeR factor R workR factor obsHighest resolutionLowest resolutionNumber reflection R freeNumber reflection obsPercent reflection R freePercent reflection obsOverall SU BOverall SU MLR Free selection detailsCross valid methodOverall ESU ROverall ESU R FreeSolvent ion probe radiiSolvent shrinkage radiiSolvent vdw probe radiiStereochemistry target valuesSolvent model details
1129.873-2.08-1.18-0.54-0.22-3.252.300.8330.841HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS0.332270.330930.331003.80146.674656877305.0100.0036.3870.487RANDOMTHROUGHOUT1.6330.5980.800.801.20MAXIMUM LIKELIHOOD WITH PHASESMASK
ELECTRON MICROSCOPY
Number of atoms included #1Total: 19015
Refine LS restraints
Refine IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.01919530
ELECTRON MICROSCOPYr_bond_other_d0.0030.02018262
ELECTRON MICROSCOPYr_angle_refined_deg1.4911.97426641
ELECTRON MICROSCOPYr_angle_other_deg1.0233.00041589
ELECTRON MICROSCOPYr_dihedral_angle_1_deg7.0945.0002379
ELECTRON MICROSCOPYr_dihedral_angle_2_deg30.79322.733849
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.16715.0003044
ELECTRON MICROSCOPYr_dihedral_angle_4_deg12.07615.000142
ELECTRON MICROSCOPYr_chiral_restr0.0990.2002900
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.02022295
ELECTRON MICROSCOPYr_gen_planes_other0.0040.0204877
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

Dom IDAuth asym IDEns IDNumberRms dev position
1A183620.08
2E183620.08
1A283660.06
2I283660.06
1B379080.07
2F379080.07
1B478960.07
2J478960.07
1C582700.07
2G582700.07
1C682640.08
2K682640.08
1D779380.08
2H779380.08
1D879940.07
2L879940.07
1E983680.08
2I983680.08
1F1078860.05
2J1078860.05
1G1184200.07
2K1184200.07
1H1280400.08
2L1280400.08
Refine LS shellHighest resolution: 3.8 Å / R factor R free: 0.539 / R factor R work: 0.55 / Lowest resolution: 3.899 Å / Number reflection R free: 355 / Number reflection R work: 6516 / Total number of bins used: 20 / Percent reflection obs: 1

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