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- EMDB-21371: Single particle reconstruction of glucose isomerase from Streptom... -

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Entry
Database: EMDB / ID: EMD-21371
TitleSingle particle reconstruction of glucose isomerase from Streptomyces rubiginosus based on data acquired in the presence of substantial aberrations
Map dataSingle particle reconstruction of glucose isomerase from Streptomyces rubiginosus based on data acquired in the presence of substantial aberrations
Sample
  • Complex: glucose isomerase
    • Protein or peptide: xylose isomerase
  • Ligand: MANGANESE (II) ION
  • Ligand: water
Keywordsglucose isomerase / ISOMERASE
Function / homology
Function and homology information


xylose isomerase / xylose isomerase activity / D-xylose metabolic process / magnesium ion binding / identical protein binding / cytoplasm
Similarity search - Function
Xylose isomerase, actinobacteria / Xylose isomerase / Xylose isomerase family profile. / : / Xylose isomerase-like, TIM barrel domain / Xylose isomerase-like TIM barrel / Xylose isomerase-like superfamily
Similarity search - Domain/homology
Biological speciesStreptomyces rubiginosus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 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 9, 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: 5
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vrs
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21371.png

Downloads & links

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Map

FileDownload / File: emd_21371.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle particle reconstruction of glucose isomerase from Streptomyces rubiginosus based on data acquired in the presence of substantial aberrations
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 256 pix.
= 232.96 Å		0.91 Å/pix.
x 256 pix.
= 232.96 Å		0.91 Å/pix.
x 256 pix.
= 232.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.0 / Movie #1: 5
Minimum - Maximum-2.951865 - 18.765633000000001
Average (Standard dev.)0.08668584 (±0.7685981)
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-2.95218.7660.087

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Supplemental data

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Sample components

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Entire : glucose isomerase

EntireName: glucose isomerase
Components
  • Complex: glucose isomerase
    • Protein or peptide: xylose isomerase
  • Ligand: MANGANESE (II) ION
  • Ligand: water

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Supramolecule #1: glucose isomerase

SupramoleculeName: glucose isomerase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Streptomyces rubiginosus (bacteria)
Molecular weightTheoretical: 172.9 KDa

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Macromolecule #1: xylose isomerase

MacromoleculeName: xylose isomerase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: xylose isomerase
Source (natural)Organism: Streptomyces rubiginosus (bacteria)
Molecular weightTheoretical: 43.283297 KDa
SequenceString: MNYQPTPEDR FTFGLWTVGW QGRDPFGDAT RRALDPVESV RRLAELGAHG VTFHDDDLIP FGSSDSEREE HVKRFRQALD DTGMKVPMA TTNLFTHPVF KDGGFTANDR DVRRYALRKT IRNIDLAVEL GAETYVAWGG REGAESGGAK DVRDALDRMK E AFDLLGEY ...String:
MNYQPTPEDR FTFGLWTVGW QGRDPFGDAT RRALDPVESV RRLAELGAHG VTFHDDDLIP FGSSDSEREE HVKRFRQALD DTGMKVPMA TTNLFTHPVF KDGGFTANDR DVRRYALRKT IRNIDLAVEL GAETYVAWGG REGAESGGAK DVRDALDRMK E AFDLLGEY VTSQGYDIRF AIEPKPNEPR GDILLPTVGH ALAFIERLER PELYGVNPEV GHEQMAGLNF PHGIAQALWA GK LFHIDLN GQNGIKYDQD LRFGAGDLRA AFWLVDLLES AGYSGPRHFD FKPPRTEDFD GVWASAAGCM RNYLILKERA AAF RADPEV QEALRASRLD ELARPTAADG LQALLDDRSA FEEFDVDAAA ARGMAFERLD QLAMDHLLGA RG

UniProtKB: Xylose isomerase

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Macromolecule #2: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 2 / Number of copies: 8 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 16 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration40 mg/mL
BufferpH: 7.5 / Component - Concentration: 20.0 mM / Component - Name: HEPES
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS TALOS
Alignment procedureBasic - Residual tilt: 5.3 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: 1-200 / Number grids imaged: 1 / Number real images: 202 / Average exposure time: 100.0 sec. / Average electron dose: 120.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 114522
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - details: Custom / Details: We used the approach implemented in cisTEM. / Number images used: 61909
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cisTEM
Final angle assignmentType: OTHER
Details: Custom software applying weighted correlation coefficients
Final 3D classificationNumber classes: 100

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Atomic model buiding 1

Initial modelPDB ID:

5vr0


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-6vrs:
Single particle reconstruction of glucose isomerase from Streptomyces rubiginosus based on data acquired in the presence of substantial aberrations

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