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- EMDB-21376: Single particle reconstruction of HemQ from Geobacillus based on ... -

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Basic information

Entry
Database: EMDB / ID: EMD-21376
TitleSingle particle reconstruction of HemQ from Geobacillus based on data acquired in the presence of substantial aberrations
Map dataThe pixel spacing corresponds to the detector pixel. The data were collected in superresolution mode so the data pixel is 0.455.
Sample
  • Complex: HemQ
    • Protein or peptide: HemQ
Keywordsoxidoreductase / heme-binding
Function / homologyCoproheme decarboxylase / Heme-dependent peroxidase ChdC/CLD / Chlorite dismutase / Dimeric alpha-beta barrel / heme biosynthetic process / peroxidase activity / heme binding / metal ion binding / Coproheme decarboxylase
Function and homology information
Biological speciesGeobacillus sp. (strain Y412MC52) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsBromberg R / Guo Y
Funding support United States, 5 items
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 11, 2020-
Header (metadata) releaseFeb 19, 2020-
Map releaseFeb 19, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.75
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.75
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vsc
  • Surface level: 0.75
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21376.png

Downloads & links

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Map

FileDownload / File: emd_21376.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe pixel spacing corresponds to the detector pixel. The data were collected in superresolution mode so the data pixel is 0.455.
Voxel sizeX=Y=Z: 0.91 Å
Density
Contour LevelBy AUTHOR: 0.75 / Movie #1: 0.75
Minimum - Maximum-1.4984062 - 3.8149874
Average (Standard dev.)0.008714601 (±0.110630356)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 232.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.910.910.91
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z232.960232.960232.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-1.4983.8150.009

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Supplemental data

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Sample components

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Entire : HemQ

EntireName: HemQ
Components
  • Complex: HemQ
    • Protein or peptide: HemQ

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Supramolecule #1: HemQ

SupramoleculeName: HemQ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Geobacillus sp. (strain Y412MC52) (bacteria)
Molecular weightTheoretical: 144 KDa

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Macromolecule #1: HemQ

MacromoleculeName: HemQ / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
EC number: Oxidoreductases; Acting on a peroxide as acceptor; Peroxidases
Source (natural)Organism: Geobacillus sp. (strain Y412MC52) (bacteria) / Strain: Y412MC52
Molecular weightTheoretical: 28.817609 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSEAAQTLDG WYCLHDFRTI DWSAWKTLPN EEREAAISEF LALVDQWETT ESEKQGSHAV YTIVGQKADI LFMILRPTLD ELHEIETAL NKTKLADYLL PAYSYVSVVE LSNYLASGSE DPYQIPEVRR RLYPILPKTN YICFYPMDKR RQGNDNWYML S MEQRRELM ...String:
MSEAAQTLDG WYCLHDFRTI DWSAWKTLPN EEREAAISEF LALVDQWETT ESEKQGSHAV YTIVGQKADI LFMILRPTLD ELHEIETAL NKTKLADYLL PAYSYVSVVE LSNYLASGSE DPYQIPEVRR RLYPILPKTN YICFYPMDKR RQGNDNWYML S MEQRRELM RAHGMTGRKY AGKVTQIITG SVGLDDFEWG VTLFSDDALQ FKKLVYEMRF DEVSARFGEF GSFFVGTRLP ME NVSSFFH V

UniProtKB: Coproheme decarboxylase

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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

Concentration28 mg/mL
BufferpH: 7.5 / Component - Concentration: 20.0 mM / Component - Name: HEPES / Details: 100 mM NaCl
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
Alignment procedureBasic - Residual tilt: 7.0 mrad
DetailsThe goal of the experiment was to show that it is possible to perform high resolution reconstruction in the presence of higher order aberrations.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-100 / Number grids imaged: 1 / Number real images: 257 / Average exposure time: 40.0 sec. / Average electron dose: 90.0 e/Å2 / Details: 173 movies were used in the reconstruction.

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Image processing

Particle selectionNumber selected: 236091
Startup modelType of model: NONE
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cisTEM
Final 3D classificationNumber classes: 100
Final angle assignmentType: OTHER
Details: Custom software applying weighted correlation coefficients
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - details: Custom
Details: There is very strong preferred orientation that generates a number of problems, in addition to high level of aberrations.
Number images used: 129446

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Atomic model buiding 1

Initial modelPDB ID:

1t0t


Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsCOOT was crucial as well.
RefinementSpace: RECIPROCAL / Protocol: OTHER / Target criteria: REFMAC
Output model

PDB-6vsc:
Single particle reconstruction of HemQ from Geobacillus based on data acquired in the presence of substantial aberrations

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