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- PDB-5ni1: CryoEM structure of haemoglobin at 3.2 A determined with the Volt... -

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Basic information

Entry
Database: PDB / ID: 5ni1
TitleCryoEM structure of haemoglobin at 3.2 A determined with the Volta phase plate
Components
  • Hemoglobin subunit alpha
  • Hemoglobin subunit beta
KeywordsOXYGEN TRANSPORT / Volta phase plate / Single particle analysis / Hemoglobin
Function / homology
Function and homology information


nitric oxide transport / hemoglobin alpha binding / haptoglobin-hemoglobin complex / organic acid binding / cellular oxidant detoxification / hemoglobin binding / renal absorption / hemoglobin complex / oxygen transport / Scavenging of heme from plasma ...nitric oxide transport / hemoglobin alpha binding / haptoglobin-hemoglobin complex / organic acid binding / cellular oxidant detoxification / hemoglobin binding / renal absorption / hemoglobin complex / oxygen transport / Scavenging of heme from plasma / endocytic vesicle lumen / blood vessel diameter maintenance / hydrogen peroxide catabolic process / oxygen carrier activity / Late endosomal microautophagy / Heme signaling / response to hydrogen peroxide / carbon dioxide transport / Erythrocytes take up oxygen and release carbon dioxide / Erythrocytes take up carbon dioxide and release oxygen / Cytoprotection by HMOX1 / platelet aggregation / oxygen binding / regulation of blood pressure / Chaperone Mediated Autophagy / positive regulation of nitric oxide biosynthetic process / tertiary granule lumen / Factors involved in megakaryocyte development and platelet production / blood microparticle / ficolin-1-rich granule lumen / iron ion binding / heme binding / Neutrophil degranulation / extracellular space / extracellular exosome / extracellular region / membrane / metal ion binding / cytosol
Similarity search - Function
Hemoglobin, pi / Hemoglobin, alpha-type / Hemoglobin, beta-type / Globin/Protoglobin / Globins / Globin family profile. / Globin-like / Globin / Globin / Globin-like superfamily ...Hemoglobin, pi / Hemoglobin, alpha-type / Hemoglobin, beta-type / Globin/Protoglobin / Globins / Globin family profile. / Globin-like / Globin / Globin / Globin-like superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Hemoglobin subunit beta / Hemoglobin subunit alpha
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsKhoshouei, M. / Radjainia, M. / Bunker, R. / Baumeister, W. / Danev, R.
Citation
Journal: Nat Commun / Year: 2017
Title: Cryo-EM structure of haemoglobin at 3.2 Å determined with the Volta phase plate.
Authors: Maryam Khoshouei / Mazdak Radjainia / Wolfgang Baumeister / Radostin Danev /
Abstract: With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way. These cameras are superior to previous detectors in coping with ...With the advent of direct electron detectors, the perspectives of cryo-electron microscopy (cryo-EM) have changed in a profound way. These cameras are superior to previous detectors in coping with the intrinsically low contrast and beam-induced motion of radiation-sensitive organic materials embedded in amorphous ice, and hence they have enabled the structure determination of many macromolecular assemblies to atomic or near-atomic resolution. Nevertheless, there are still limitations and one of them is the size of the target structure. Here, we report the use of a Volta phase plate in determining the structure of human haemoglobin (64 kDa) at 3.2 Å. Our results demonstrate that this method can be applied to complexes that are significantly smaller than those previously studied by conventional defocus-based approaches. Cryo-EM is now close to becoming a fast and cost-effective alternative to crystallography for high-resolution protein structure determination.
#1: Journal: Elife / Year: 2016
Title: Cryo-EM single particle analysis with the Volta phase plate.
Authors: Radostin Danev / Wolfgang Baumeister /
Abstract: We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining ...We present a method for in-focus data acquisition with a phase plate that enables near-atomic resolution single particle reconstructions. Accurate focusing is the determining factor for obtaining high quality data. A double-area focusing strategy was implemented in order to achieve the required precision. With this approach we obtained a 3.2 Å resolution reconstruction of the Thermoplasma acidophilum 20S proteasome. The phase plate matches or slightly exceeds the performance of the conventional defocus approach. Spherical aberration becomes a limiting factor for achieving resolutions below 3 Å with in-focus phase plate images. The phase plate could enable single particle analysis of challenging samples in terms of small size, heterogeneity and flexibility that are difficult to solve by the conventional defocus approach.
History
DepositionMar 22, 2017Deposition site: PDBE / Processing site: PDBE
SupersessionApr 12, 2017ID: 5ME2
Revision 1.0Apr 12, 2017Provider: repository / Type: Initial release
Revision 1.1Oct 17, 2018Group: Data collection / Other / Refinement description / Category: cell / em_image_scans / refine / Item: _cell.Z_PDB
Revision 1.2Dec 11, 2019Group: Database references / Other / Category: atom_sites / citation / citation_author
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Assembly

Deposited unit
A: Hemoglobin subunit alpha
B: Hemoglobin subunit beta
C: Hemoglobin subunit alpha
D: Hemoglobin subunit beta
hetero molecules


Theoretical massNumber of molelcules
Total (without water)64,5478
Polymers62,0814
Non-polymers2,4664
Water43224
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21C
12B
22D

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1111A1 - 141
2111C1 - 141
1121B1 - 146
2121D1 - 146

NCS ensembles :
ID
1
2

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-1, -0.000463, -0.00017), (0.000463, -1, -0.000505), (-0.00017, -0.000505, 1)105.04034, 105.01142, 0.03988

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Components

#1: Protein Hemoglobin subunit alpha / / Alpha-globin / Hemoglobin alpha chain


Mass: 15150.353 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HBA1, HBA2 / Production host: Homo sapiens (human) / References: UniProt: P69905
#2: Protein Hemoglobin subunit beta / / Beta-globin / Hemoglobin beta chain


Mass: 15890.198 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HBB / Production host: Homo sapiens (human) / References: UniProt: P68871
#3: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Formula: H2O

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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: Hemoglobin / Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE-PROPANE / Humidity: 100 %

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 175300 / Symmetry type: POINT
RefinementResolution: 3.2→105 Å / Cor.coef. Fo:Fc: 0.574 / SU B: 18.168 / SU ML: 0.278 / ESU R: 0.34
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.37077 --
obs0.37077 74004 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 60.032 Å2
Baniso -1Baniso -2Baniso -3
1--1.39 Å2-1.67 Å2-0.01 Å2
2--0.67 Å2-0.01 Å2
3---0.72 Å2
Refinement stepCycle: 1 / Total: 4580
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.010.0194703
ELECTRON MICROSCOPYr_bond_other_d0.0020.024331
ELECTRON MICROSCOPYr_angle_refined_deg1.53226449
ELECTRON MICROSCOPYr_angle_other_deg1.296310003
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.7125570
ELECTRON MICROSCOPYr_dihedral_angle_2_deg33.07723.889180
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.44615705
ELECTRON MICROSCOPYr_dihedral_angle_4_deg11.3571512
ELECTRON MICROSCOPYr_chiral_restr0.1040.2708
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.0215272
ELECTRON MICROSCOPYr_gen_planes_other0.0030.02984
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_it5.5645.6852292
ELECTRON MICROSCOPYr_mcbond_other5.525.6732291
ELECTRON MICROSCOPYr_mcangle_it9.8188.4842858
ELECTRON MICROSCOPYr_mcangle_other9.8338.4962859
ELECTRON MICROSCOPYr_scbond_it7.1026.4952411
ELECTRON MICROSCOPYr_scbond_other7.0916.4922409
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other12.7439.2523591
ELECTRON MICROSCOPYr_long_range_B_refined18.71764.9355272
ELECTRON MICROSCOPYr_long_range_B_other18.72164.9435273
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: tight thermal / Weight position: 0.5

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11A10680.34
22B11230.12
LS refinement shellResolution: 3.2→3.283 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.965 5475 -
Rfree-0 -
obs--100 %

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