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- EMDB-27902: MicroED structure of triclinic lysozyme recorded on K3 -

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Basic information

Entry
Database: EMDB / ID: EMD-27902
TitleMicroED structure of triclinic lysozyme recorded on K3
Map data2mFo-DFc
Sample
  • Complex: Lysozyme
    • Protein or peptide: Lysozyme C
  • Ligand: NITRATE IONNitrate
  • Ligand: water
Function / homology
Function and homology information


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 ...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 / Nannenga BL / 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 / Year: 2022
Title: Electron-counting MicroED data with the K2 and K3 direct electron detectors.
Authors: Max T B Clabbers / Michael W Martynowycz / Johan Hattne / Brent L Nannenga / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances ...Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.
History
DepositionAug 19, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateOct 19, 2022-
Current statusOct 19, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27902.png

Downloads & links

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Map

FileDownload / File: emd_27902.map.gz / Format: CCP4 / Size: 25 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2mFo-DFc
Voxel sizeX: 0.2931 Å / Y: 0.2848 Å / Z: 0.275 Å
Density
Contour LevelBy AUTHOR: 1.0
Minimum - Maximum-2.2371824 - 12.134751
Average (Standard dev.)-0.0053252387 (±0.9649187)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin-128-145-123
Dimensions172218175
Spacing175172218
CellA: 51.2925 Å / B: 48.9856 Å / C: 59.95 Å
α: 87.851 ° / β: 108.849 ° / γ: 112.6 °

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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 / Chimera: Yes / 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: 16.25766 KDa
SequenceString:
MRSLLILVLC FLPLAALGKV FGRCELAAAM KRHGLDNYRG YSLGNWVCAA KFESNFNTQA TNRNTDGSTD YGILQINSRW WCNDGRTPG SRNLCNIPCS ALLSSDITAS VNCAKKIVSD GNGMNAWVAW RNRCKGTDVQ AWIRGCRL

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Macromolecule #2: NITRATE ION

MacromoleculeName: NITRATE ION / type: ligand / ID: 2 / Number of copies: 1 / 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: 118 / 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: 373 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: GATAN K3 (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Digitization - Sampling interval: 5.0 µm / Number grids imaged: 1 / Number real images: 1 / Number diffraction images: 5600 / Average exposure time: 0.1 sec. / Average electron dose: 0.001 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Crystal parametersUnit cell - A: 26.38 Å / Unit cell - B: 30.76 Å / Unit cell - C: 33 Å / Unit cell - γ: 112.60 ° / Unit cell - α: 87.85 ° / Unit cell - β: 108.85 ° / Space group: P-1
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

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL / Overall B value: 12.94 / Target criteria: Maximum likelihood
Output model

PDB-8e54:
MicroED structure of triclinic lysozyme recorded on K3

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