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- EMDB-21203: 1.4A damaged structure of GSNQNNF used to determine initial phase... -

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Basic information

Entry
Database: EMDB / ID: EMD-21203
Title1.4A damaged structure of GSNQNNF used to determine initial phases from radiation damage
Map data2mFo-Fc map for the damaged GSNQNNF structure refined from the solved low-dose structure that had initially determined experimental phases
Sample
  • Complex: Synthetic proto-filament
    • Protein or peptide: GSNQNNF
  • Ligand: ZINC ION
  • Ligand: ACETATE ION
  • Ligand: water
KeywordsMicroED / damage / phasing / RIP / protein fibril
Biological speciessynthetic construct (others)
Methodelectron crystallography / cryo EM
AuthorsMartynowycz MW / Hattne J
CitationJournal: Structure / Year: 2020
Title: Experimental Phasing of MicroED Data Using Radiation Damage.
Authors: Michael W Martynowycz / Johan Hattne / Tamir Gonen /
Abstract: We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present ...We previously demonstrated that microcrystal electron diffraction (MicroED) can be used to determine atomic-resolution structures from vanishingly small three-dimensional crystals. Here, we present an example of an experimentally phased structure using only MicroED data. The structure of a seven-residue peptide is solved starting from differences to the diffraction intensities induced by structural changes due to radiation damage. The same wedge of reciprocal space was recorded twice by continuous-rotation MicroED from a set of 11 individual crystals. The data from the first pass were merged to make a "low-dose dataset." The data from the second pass were similarly merged to form a "damaged dataset." Differences between these two datasets were used to identify a single heavy-atom site from a Patterson difference map, and initial phases were generated. Finally, the structure was completed by iterative cycles of modeling and refinement.
History
DepositionJan 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: 0.35997
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.35997
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vhc
  • Surface level: 0.35997
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vhc
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21203.png

Downloads & links

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Map

FileDownload / File: emd_21203.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2mFo-Fc map for the damaged GSNQNNF structure refined from the solved low-dose structure that had initially determined experimental phases
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.44 Å/pix.
x 93 pix.
= 40.966 Å		0.41 Å/pix.
x 101 pix.
= 41.073 Å		0.44 Å/pix.
x 93 pix.
= 41.182 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.40667 Å / Y: 0.44281 Å / Z: 0.4405 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.35997 / Movie #1: 0.35997
Minimum - Maximum-0.8414527 - 1.840897
Average (Standard dev.)0.0012663071 (±0.23998328)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-532-20
Dimensions1019393
Spacing1019393
CellA: 41.073334 Å / B: 41.181564 Å / C: 40.9665 Å
α: 83.6 ° / β: 84.98 ° / γ: 83.31 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.406663366336630.442817204301080.44049462365591
M x/y/z1019393
origin x/y/z0.0000.0000.000
length x/y/z41.07341.18240.966
α/β/γ83.60084.98083.310
start NX/NY/NZ-532-20
NX/NY/NZ1019393
MAP C/R/S213
start NC/NR/NS2-53-20
NC/NR/NS9310193
D min/max/mean-0.8411.8410.001

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Supplemental data

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Sample components

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Entire : Synthetic proto-filament

EntireName: Synthetic proto-filament
Components
  • Complex: Synthetic proto-filament
    • Protein or peptide: GSNQNNF
  • Ligand: ZINC ION
  • Ligand: ACETATE ION
  • Ligand: water

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Supramolecule #1: Synthetic proto-filament

SupramoleculeName: Synthetic proto-filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 899.141 Da

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Macromolecule #1: GSNQNNF

MacromoleculeName: GSNQNNF / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 779.756 Da
SequenceString:
GSNQNNF

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Macromolecule #2: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #3: ACETATE ION

MacromoleculeName: ACETATE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: ACT
Molecular weightTheoretical: 59.044 Da
Chemical component information

ChemComp-ACT:
ACETATE ION

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 1 / 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
Processingelectron crystallography
Aggregation state3D array

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Sample preparation

Concentration10 mg/mL
BufferpH: 6
Component:
ConcentrationFormulaName
0.1 MC6H13NO4SMES
10.0 %C6H14O2MPD
GridModel: Quantifoil R2/4 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 30 % / Instrument: FEI VITROBOT MARK IV
DetailsHanging drop.

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number grids imaged: 1 / Number real images: 736 / Number diffraction images: 736 / Average exposure time: 2.1 sec. / Average electron dose: 0.00588 e/Å2 / Details: Images collected as a movies.
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 730 mm
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsRolling shutter and binned by 2.
Final reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES
Crystallography statisticsNumber intensities measured: 6314 / Number structure factors: 722 / Fourier space coverage: 78.1 / R sym: 0.21 / R merge: 0.199 / Overall phase error: 26 / Overall phase residual: 26 / Phase error rejection criteria: 0 / High resolution: 1.4 Å
Details: Model from the low-dose set refined against the damage dataset without any changes.
Shell - Shell ID: 1 / Shell - High resolution: 1.4 Å / Shell - Low resolution: 13.97 Å / Shell - Number structure factors: 722 / Shell - Phase residual: 26 / Shell - Fourier space coverage: 78.1 / Shell - Multiplicity: 8.7

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Atomic model buiding 1

Initial modelPDB ID:

6clj


Chain - Chain ID: A / Chain - Residue range: 1-7 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 5
Output model

PDB-6vhc:
1.4A damaged structure of GSNQNNF used to determine initial phases from radiation damage

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