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- PDB-6lav: MicroED structure of lysozyme at 1.73A determained using crystal ... -

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Basic information

Entry
Database: PDB / ID: 6lav
TitleMicroED structure of lysozyme at 1.73A determained using crystal lamellas prepared by focused ion beam milling
ComponentsLysozyme C
KeywordsHYDROLASE / Lysozyme / MicroED / Focused Ion Beam / crystal lamella
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
Lysozyme - #10 / 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 / Lysozyme-like domain superfamily ...Lysozyme - #10 / 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 / Lysozyme-like domain superfamily / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
ACETATE ION / Lysozyme C
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.73 Å
AuthorsZhou, H. / Luo, Z. / Li, X.
Funding support China, 4items
OrganizationGrant numberCountry
National Natural Science Foundation of China31570730 China
National Natural Science Foundation of China31722015 China
Ministry of Science and Technology (China)2016YFA0501102 China
Ministry of Science and Technology (China)2016YFA0501902 China
CitationJournal: J Struct Biol / Year: 2019
Title: Using focus ion beam to prepare crystal lamella for electron diffraction.
Authors: Heng Zhou / Zhipu Luo / Xueming Li /
Abstract: Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the ...Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the electron crystallographic method on large protein crystals with micrometer or larger sizes. Here, we used the focused ion beam (FIB) equipped on the scanning electron microscope (SEM) to mill a large crystal to thin lamella. The influences of the milling on the crystal lamella were observed and investigated, including radiation damage on the crystal surface during the FIB imaging, deformation of the thin crystal lamella, and variation in the diffraction intensities under electron radiation. These observations provide important information to optimize the FIB milling, and hence is important to obtain high-quality crystal samples for routine structure determination of protein crystals using the electron cryo-microscope.
History
DepositionNov 13, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 27, 2019Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Lysozyme C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,4493
Polymers14,3311
Non-polymers1182
Water66737
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area420 Å2
ΔGint-1 kcal/mol
Surface area6760 Å2
Unit cell
Length a, b, c (Å)78.986, 78.986, 37.889
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein Lysozyme C / 1 / 4-beta-N-acetylmuramidase C / Allergen Gal d IV


Mass: 14331.160 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / References: UniProt: P00698, lysozyme
#2: Chemical ChemComp-ACT / ACETATE ION / Acetate


Mass: 59.044 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C2H3O2
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 37 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: lysozyme / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Gallus gallu (chicken)
Buffer solutionpH: 4.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 0.057 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
EM diffractionCamera length: 1200 mm
EM diffraction shellResolution: 1.7→1.76 Å / Fourier space coverage: 79.1 % / Multiplicity: 9.7 / Num. of structure factors: 1002 / Phase residual: 1 °
EM diffraction statsFourier space coverage: 89 % / High resolution: 1.7 Å / Num. of intensities measured: 121300 / Num. of structure factors: 11548 / Phase error: 0 ° / Phase residual: 1 ° / Phase error rejection criteria: 60 / Rmerge: 0.281 / Rsym: 0.281
DetectorDate: Feb 2, 2018

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0222refinement
PDB_EXTRACT3.25data extraction
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 78.99 Å / B: 78.99 Å / C: 37.89 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 1.73 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL
RefinementResolution: 1.73→14.67 Å / Cor.coef. Fo:Fc: 0.94 / Cor.coef. Fo:Fc free: 0.924 / SU B: 3.69 / SU ML: 0.113 / Cross valid method: THROUGHOUT / ESU R: 0.16 / ESU R Free: 0.147
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2575 550 4.8 %RANDOM
Rwork0.2203 ---
obs0.2221 10947 88.68 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso max: 90.95 Å2 / Biso mean: 28.358 Å2 / Biso min: 12.1 Å2
Baniso -1Baniso -2Baniso -3
1-0.1 Å2-0 Å2-0 Å2
2--0.1 Å2-0 Å2
3----0.2 Å2
Refinement stepCycle: final / Resolution: 1.73→14.67 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1001 0 8 37 1046
Biso mean--41.56 28.76 -
Num. residues----129
LS refinement shellResolution: 1.732→1.777 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.281 25 -
Rwork0.326 679 -
all-704 -
obs--76.77 %

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