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- EMDB-26596: MicroED structure of triclinic lysozyme -

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Basic information

Entry
Database: EMDB / ID: EMD-26596
TitleMicroED structure of triclinic lysozyme
Map data
Sample
  • Complex: Lysozyme
    • Protein or peptide: Lysozyme C
  • Ligand: NITRATE IONNitrate
  • 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 / killing of cells of another organism / defense response to Gram-negative bacterium / defense response to Gram-positive bacterium ...Lactose synthesis / Antimicrobial peptides / Neutrophil degranulation / beta-N-acetylglucosaminidase activity / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / killing of cells of another organism / defense response to Gram-negative bacterium / 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
AuthorsClabbers MTB / Martynowycz MW / 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)GM136508 United States
CitationJournal: J Struct Biol X / Year: 2022
Title: Hydrogens and hydrogen-bond networks in macromolecular MicroED data.
Authors: Max T B Clabbers / Michael W Martynowycz / Johan Hattne / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray ...Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray crystallography. Electrons interact with the electrostatic potential of the sample, which means that the scattered electrons carry information about the charged state of atoms and provide relatively stronger contrast for visualizing hydrogen atoms. Accurately identifying the positions of hydrogen atoms, and by extension the hydrogen bonding networks, is of importance for understanding protein structure and function, in particular for drug discovery. However, identification of individual hydrogen atom positions typically requires atomic resolution data, and has thus far remained elusive for macromolecular MicroED. Recently, we presented the structure of triclinic hen egg-white lysozyme at 0.87 Å resolution. The corresponding data were recorded under low exposure conditions using an electron-counting detector from thin crystalline lamellae. Here, using these subatomic resolution MicroED data, we identified over a third of all hydrogen atom positions based on strong difference peaks, and directly visualize hydrogen bonding interactions and the charged states of residues. Furthermore, we find that the hydrogen bond lengths are more accurately described by the inter-nuclei distances than the centers of mass of the corresponding electron clouds. We anticipate that MicroED, coupled with ongoing advances in data collection and refinement, can open further avenues for structural biology by uncovering the hydrogen atoms and hydrogen bonding interactions underlying protein structure and function.
History
DepositionApr 5, 2022-
Header (metadata) releaseMar 15, 2023-
Map releaseMar 15, 2023-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26596.png

Downloads & links

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Map

FileDownload / File: emd_26596.map.gz / Format: CCP4 / Size: 63.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX: 0.21136 Å / Y: 0.21333 Å / Z: 0.20631 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-2.3125014 - 15.775169
Average (Standard dev.)-0.0020035517 (±0.96471184)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-168-131-109
Dimensions249297224
Spacing224249297
CellA: 47.344643 Å / B: 53.12 Å / C: 61.27481 Å
α: 88.319 ° / β: 109.095 ° / γ: 112.075 °

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Supplemental data

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Sample components

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Entire : Lysozyme

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

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Supramolecule #1: Lysozyme

SupramoleculeName: Lysozyme / type: complex / ID: 1 / Chimera: Yes / 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 / Nitrate

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 112 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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: 4.5
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 10.0 nm
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: LEICA PLUNGER
DetailsProtein crystal lamellae

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Electron microscopy

MicroscopeFEI TITAN KRIOS
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: 1536 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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 / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

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
Final reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: REFMAC (ver. 5.8.0267)
Merging software listSoftware - Name: AIMLESS
DetailsBinned by 2

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 11.981 / Target criteria: Maximum likelihood
Output model

PDB-7uly:
MicroED structure of triclinic lysozyme

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