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- PDB-6vsa: Single particle reconstruction of HemQ from Geobacillus based on ... -

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Basic information

Entry
Database: PDB / ID: 6vsa
TitleSingle particle reconstruction of HemQ from Geobacillus based on data acquired in the presence of substantial aberrations
ComponentsHemQ
KeywordsOXIDOREDUCTASE / heme-binding
Function / homology
Function and homology information


heme biosynthetic process / peroxidase activity / heme binding / metal ion binding
Similarity search - Function
Apc35880; domain 1 / Coproheme decarboxylase / Heme-dependent peroxidase ChdC/CLD / Chlorite dismutase / Dimeric alpha-beta barrel / Alpha-Beta Plaits / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
Coproheme decarboxylase
Similarity search - Component
Biological speciesGeobacillus sp. (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.32 Å
AuthorsBromberg, R. / Guo, Y. / Borek, D. / Otwinowski, Z.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM117080 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM118619 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R21GM126406 United States
Department of Energy (DOE, United States)DE-SC0019600 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)HHSN272201700060C United States
Citation
Journal: IUCrJ / Year: 2020
Title: High-resolution cryo-EM reconstructions in the presence of substantial aberrations.
Authors: Raquel Bromberg / Yirui Guo / Dominika Borek / Zbyszek Otwinowski /
Abstract: Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula ...Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula quantifying information loss owing to their presence is inferred that explains why Fourier-shell coefficient-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. The analysis is validated with reference-based aberration refinement for two cryo-EM SPR data sets acquired with a 200 kV microscope in the presence of coma exceeding 40 µm, and 2.3 and 2.7 Å reconstructions for 144 and 173 kDa particles, respectively, were obtained. The results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher order aberrations, and address inconsistent guidelines regarding the level of aberrations that is acceptable in cryo-EM SPR experiments.
#1: Journal: Biorxiv / Year: 2020
Title: High-resolution cryo-EM reconstructions in the presence of substantial aberrations
Authors: Bromberg, R. / Guo, Y. / Borek, D. / Otwinowski, Z.
History
DepositionFeb 10, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 19, 2020Provider: repository / Type: Initial release
Revision 1.1May 13, 2020Group: Database references / Category: citation / citation_author
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _citation.journal_id_ISSN / _database_2.pdbx_DOI ..._citation.journal_id_ISSN / _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _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
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-21373
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
V: HemQ
W: HemQ
X: HemQ
Y: HemQ
Z: HemQ


Theoretical massNumber of molelcules
Total (without water)144,0885
Polymers144,0885
Non-polymers00
Water75742
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16520 Å2
ΔGint-60 kcal/mol
Surface area43060 Å2
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11V
21W
12V
22X
13V
23Y
14V
24Z
15W
25X
16W
26Y
17W
27Z
18X
28Y
19X
29Z
110Y
210Z

NCS domain segments:

Component-ID: _ / Beg auth comp-ID: ALA / Beg label comp-ID: ALA / End auth comp-ID: VAL / End label comp-ID: VAL / Refine code: _ / Auth seq-ID: 4 - 248 / Label seq-ID: 4 - 248

Dom-IDEns-IDAuth asym-IDLabel asym-ID
11VA
21WB
12VA
22XC
13VA
23YD
14VA
24ZE
15WB
25XC
16WB
26YD
17WB
27ZE
18XC
28YD
19XC
29ZE
110YD
210ZE

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

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Components

#1: Protein
HemQ / UPF0447 protein GYMC52_3505


Mass: 28817.609 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacillus sp. (strain Y412MC52) (bacteria)
Strain: Y412MC52 / Gene: GYMC52_3505 / Plasmid: pMCSG7 / Production host: Escherichia coli (E. coli)
References: UniProt: A0A0E0TGF4, Oxidoreductases; Acting on a peroxide as acceptor; Peroxidases
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 42 / 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: HemQ / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.144 MDa / Experimental value: YES
Source (natural)Organism: Geobacillus sp. (strain Y412MC52) (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5 / Details: 100 mM NaCl
Buffer componentConc.: 20 mM / Name: HEPES
SpecimenConc.: 28 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Details: The goal of the experiment was to show that it is possible to perform high resolution reconstruction in the presence of higher order aberrations.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Residual tilt: 6.9 mradians
Image recordingAverage exposure time: 40 sec. / Electron dose: 120 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 268 / Details: 258 movies were used in the reconstruction.
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 100 / Used frames/image: 1-100

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Processing

SoftwareName: REFMAC / Version: 5.8.0257 / Classification: refinement
EM software
IDNameCategory
1cisTEMparticle selection
2SerialEMimage acquisition
4cisTEMCTF correction
7MOLREPmodel fitting
9cisTEMinitial Euler assignment
13REFMACmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 156210
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 81302 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL / Target criteria: REFMAC / Details: COOT was crucial as well.
Atomic model buildingPDB-ID: 1T0T

1t0t


Accession code: 1T0T / Source name: PDB / Type: experimental model
RefinementResolution: 2.32→98.64 Å / Cor.coef. Fo:Fc: 0.979 / SU B: 7.012 / SU ML: 0.144 / ESU R: 0.196
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.24062 --
obs0.24062 117718 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 88.717 Å2
Baniso -1Baniso -2Baniso -3
1--0.03 Å20.02 Å20.02 Å2
2--0.04 Å20.02 Å2
3----0.01 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0120.0129920
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg2.0741.63813425
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg6.79451165
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.77221.565575
ELECTRON MICROSCOPYr_dihedral_angle_3_deg19.637151695
ELECTRON MICROSCOPYr_dihedral_angle_4_deg16.5961575
ELECTRON MICROSCOPYr_chiral_restr0.1380.21240
ELECTRON MICROSCOPYr_gen_planes_refined0.0120.027650
ELECTRON MICROSCOPYr_gen_planes_other
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_it14.1967.4574690
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it18.5311.3345845
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it17.999.3845230
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined31.10645342
ELECTRON MICROSCOPYr_long_range_B_other
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)
11V177040.06
12W177040.06
21V178140.06
22X178140.06
31V176680.07
32Y176680.07
41V177340.07
42Z177340.07
51W177260.06
52X177260.06
61W176860.06
62Y176860.06
71W177440.05
72Z177440.05
81X177080.07
82Y177080.07
91X177800.06
92Z177800.06
101Y177060.06
102Z177060.06
LS refinement shellResolution: 2.32→2.38 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.605 8664 -
obs--100 %

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