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- EMDB-25184: Ab initio structure of triclinic lysozyme from electron-counted M... -

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Basic information

Entry
Database: EMDB / ID: EMD-25184
TitleAb initio structure of triclinic lysozyme from electron-counted MicroED data
Map dataAb initio normalized structure factor map of P1 lysozyme after fragment placement and density modification from electron-counted MicroED data
Sample
  • Complex: Lysozyme
    • Protein or peptide: Lysozyme C
  • Ligand: NITRATE ION
  • Ligand: water
Function / homology
Function and homology information


Lactose synthesis / Antimicrobial peptides / Neutrophil degranulation / beta-N-acetylglucosaminidase activity / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / defense response to Gram-negative bacterium / killing of cells of another organism / defense response to Gram-positive bacterium ...Lactose synthesis / Antimicrobial peptides / Neutrophil degranulation / beta-N-acetylglucosaminidase activity / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / defense response to Gram-negative bacterium / killing of cells of another organism / defense response to Gram-positive bacterium / defense response to bacterium / endoplasmic reticulum / extracellular space / identical protein binding / cytoplasm
Similarity search - Function
Glycoside hydrolase, family 22, lysozyme / Glycoside hydrolase family 22 domain / Glycosyl hydrolases family 22 (GH22) domain signature. / Glycoside hydrolase, family 22 / C-type lysozyme/alpha-lactalbumin family / Glycosyl hydrolases family 22 (GH22) domain profile. / Alpha-lactalbumin / lysozyme C / Lysozyme-like domain superfamily
Similarity search - Domain/homology
Biological speciesGallus gallus (chicken) / Chicken (chicken)
Methodelectron crystallography / cryo EM
AuthorsMartynowycz MW / Clabbers MTB / Hattne J / Gonen T
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
CitationJournal: Nat Methods / Year: 2022
Title: Ab initio phasing macromolecular structures using electron-counted MicroED data.
Authors: Michael W Martynowycz / Max T B Clabbers / Johan Hattne / Tamir Gonen /
Abstract: Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals ...Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data.
History
DepositionOct 21, 2021-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25184.png

Downloads & links

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Map

FileDownload / File: emd_25184.map.gz / Format: CCP4 / Size: 11 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAb initio normalized structure factor map of P1 lysozyme after fragment placement and density modification from electron-counted MicroED data
Voxel sizeX: 0.21136 Å / Y: 0.21333 Å / Z: 0.20631 Å
Density
Contour LevelBy AUTHOR: 2.0
Minimum - Maximum-4.8827868 - 28.633507
Average (Standard dev.)3.6253355e-12 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions144160125
Spacing125144160
CellA: 26.42 Å / B: 30.72 Å / C: 33.01 Å
α: 88.319 ° / β: 109.095 ° / γ: 112.075 °

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Supplemental data

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Additional map: 2mFo-dFc map of P1 lysozyme determined ab initio...

Fileemd_25184_additional_1.map
Annotation2mFo-dFc map of P1 lysozyme determined ab initio by electron counted MicroED data
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: mFo-dFc map of P1 lysozyme determined ab initio...

Fileemd_25184_additional_2.map
AnnotationmFo-dFc map of P1 lysozyme determined ab initio by electron counted MicroED data
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Lysozyme

EntireName: Lysozyme
Components
  • Complex: Lysozyme
    • Protein or peptide: Lysozyme C
  • Ligand: NITRATE ION
  • Ligand: water

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Supramolecule #1: Lysozyme

SupramoleculeName: Lysozyme / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 14.4 KDa

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Macromolecule #1: Lysozyme C

MacromoleculeName: Lysozyme C / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: lysozyme
Source (natural)Organism: Chicken (chicken)
Molecular weightTheoretical: 14.33116 KDa
SequenceString:
KVFGRCELAA AMKRHGLDNY RGYSLGNWVC AAKFESNFNT QATNRNTDGS TDYGILQINS RWWCNDGRTP GSRNLCNIPC SALLSSDIT ASVNCAKKIV SDGNGMNAWV AWRNRCKGTD VQAWIRGCRL

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Macromolecule #2: NITRATE ION

MacromoleculeName: NITRATE ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: NO3
Molecular weightTheoretical: 62.005 Da
Chemical component information

ChemComp-NO3:
NITRATE ION

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 156 / 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

Concentration5 mg/mL
BufferpH: 4.7
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10.0 nm / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER
DetailsMilled microcrystals

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 77.0 K / Max: 90.0 K
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Digitization - Sampling interval: 28.0 µm / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 840 / Average exposure time: 0.5 sec. / Average electron dose: 0.001 e/Å2
Details: 0.15 degrees per second, 0.5 second readout, 30 to -30 degrees
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Cs: 2.7 mm / Camera length: 1202 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN / Tilt angle: -30.0, 30.0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsBinned by 2
Final reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: REFMAC
Crystal parametersUnit cell - A: 26.420 Å / Unit cell - B: 30.720 Å / Unit cell - C: 33.010 Å / Unit cell - γ: 112.075 ° / Unit cell - α: 88.319 ° / Unit cell - β: 109.095 ° / Space group: P 1
Merging software listSoftware - Name: AIMLESS
Crystallography statisticsNumber intensities measured: 569407 / Number structure factors: 64974 / Fourier space coverage: 87.58 / R sym: 0.073 / R merge: 0.236 / Overall phase error: 30 / Overall phase residual: 0 / Phase error rejection criteria: None / High resolution: 0.87 Å / Shell - Shell ID: 1 / Shell - High resolution: 0.87 Å / Shell - Low resolution: 0.9 Å / Shell - Number structure factors: 2783 / Shell - Phase residual: 30 / Shell - Fourier space coverage: 37.64 / Shell - Multiplicity: 2.1

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 12.188 / Target criteria: Maximum likelihood
Output model

PDB-7skw:
Ab initio structure of triclinic lysozyme from electron-counted MicroED data

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