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- PDB-6hu5: STRUCTURE OF HEWL BY ELECTRON DIFFRACTION AND MICROFOCUS DIFFRACTION -

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Basic information

Entry
Database: PDB / ID: 6hu5
TitleSTRUCTURE OF HEWL BY ELECTRON DIFFRACTION AND MICROFOCUS DIFFRACTION
ComponentsLysozyme C
KeywordsHYDROLASE / LYSOZYME / HEWL / ED / ELECTRON / DIFFRACTION / CRYSTAL / CHLORIDE / HALOGEN / PROTEIN / DIMER / MICROFOCUS
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
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
Biological speciesGallus gallus (chicken)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2.8 Å
AuthorsGarau, G.
CitationJournal: IUCrJ / Year: 2019
Title: Nanobeam precession-assisted 3D electron diffraction reveals a new polymorph of hen egg-white lysozyme.
Authors: Arianna Lanza / Eleonora Margheritis / Enrico Mugnaioli / Valentina Cappello / Gianpiero Garau / Mauro Gemmi /
Abstract: Recent advances in 3D electron diffraction have allowed the structure determination of several model proteins from submicrometric crystals, the unit-cell parameters and structures of which could be ...Recent advances in 3D electron diffraction have allowed the structure determination of several model proteins from submicrometric crystals, the unit-cell parameters and structures of which could be immediately validated by known models previously obtained by X-ray crystallography. Here, the first new protein structure determined by 3D electron diffraction data is presented: a previously unobserved polymorph of hen egg-white lysozyme. This form, with unit-cell parameters = 31.9, = 54.4, = 71.8 Å, β = 98.8°, grows as needle-shaped submicrometric crystals simply by vapor diffusion starting from previously reported crystallization conditions. Remarkably, the data were collected using a low-dose stepwise experimental setup consisting of a precession-assisted nanobeam of ∼150 nm, which has never previously been applied for solving protein structures. The crystal structure was additionally validated using X-ray synchrotron-radiation sources by both powder diffraction and single-crystal micro-diffraction. 3D electron diffraction can be used for the structural characterization of submicrometric macromolecular crystals and is able to identify novel protein polymorphs that are hardly visible in conventional X-ray diffraction experiments. Additionally, the analysis, which was performed on both nanocrystals and microcrystals from the same crystallization drop, suggests that an integrated view from 3D electron diffraction and X-ray microfocus diffraction can be applied to obtain insights into the molecular dynamics during protein crystal growth.
History
DepositionOct 5, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 27, 2019Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jul 17, 2019Group: Data collection / Derived calculations
Category: pdbx_struct_assembly / pdbx_struct_assembly_gen / pdbx_struct_assembly_prop

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Assembly

Deposited unit
A: Lysozyme C
B: Lysozyme C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,7695
Polymers28,6622
Non-polymers1063
Water00
1
A: Lysozyme C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,3672
Polymers14,3311
Non-polymers351
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Lysozyme C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,4023
Polymers14,3311
Non-polymers712
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)31.849, 54.380, 71.788
Angle α, β, γ (deg.)90.00, 98.82, 90.00
Int Tables number4
Space group name H-MP1211

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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.: 2 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / References: UniProt: P00698, lysozyme
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl

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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: HWEL / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Gallus gallus (chicken)
Buffer solutionpH: 7
Buffer componentConc.: 1.5 mg/mL / Name: Sodium Chloride / Formula: NaCl
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE
CrystalDensity Matthews: 2.07 Å3/Da / Density % sol: 40.5 %
Crystal growDetails: 1.5 M SODIUM CHLORIDE

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Data collection

MicroscopyModel: ZEISS LIBRA120PLUS
Electron gunElectron source: LAB6 / Accelerating voltage: 120 kV / Illumination mode: OTHER
Electron lensMode: DARK FIELD
Image recordingElectron dose: 9 e/Å2 / Film or detector model: OTHER
EM diffractionCamera length: 800 mm
EM diffraction shellResolution: 2.8→2.93 Å / Fourier space coverage: 67.1 % / Multiplicity: 2.8 / Num. of structure factors: 2993 / Phase residual: 30 °
EM diffraction statsFourier space coverage: 90 % / High resolution: 2.8 Å / Num. of intensities measured: 10909 / Num. of structure factors: 3905 / Phase error: 78 ° / Phase residual: 30.5 ° / Phase error rejection criteria: NULL / Rmerge: 0.655 / Rsym: 0.655
DiffractionMean temperature: 100 K
Diffraction sourceSource: LaB6 THERMOIONIC SOURCE / Wavelength: 0.0335
DetectorType: ASI / Detector: OTHER / Date: Oct 1, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthWavelength: 0.0335 Å / Relative weight: 1
ReflectionResolution: 2.8→42.67 Å / Num. obs: 3906 / % possible obs: 66.5 % / Redundancy: 2.8 % / Rmerge(I) obs: 0.655 / Net I/σ(I): 1.6
Reflection shellResolution: 2.8→2.97 Å / Redundancy: 2.8 % / Rmerge(I) obs: 0.839 / Mean I/σ(I) obs: 0.8 / % possible all: 67.1

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Processing

Software
NameVersionClassification
XDSdata reduction
Aimlessdata scaling
XDSphasing
CCP4phasing
PHENIX(1.13_2998)refinement
EM software
IDNameVersionCategory
6PHENIX1.13model fitting
8PHENIX1.13molecular replacement
13PHENIX1.13model refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 98.82 ° / ∠γ: 90 ° / A: 31.849 Å / B: 54.38 Å / C: 71.788 Å / Space group name: P1211 / Space group num: 4
CTF correctionType: NONE
3D reconstructionResolution: 2.8 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 10.8 / Protocol: OTHER / Target criteria: CORRELATION COEF
Atomic model buildingPDB-ID: 1B2K

1b2k

RefinementResolution: 2.8→42.67 Å / SU ML: 0.51 / Cross valid method: NONE / σ(F): 1.34 / Phase error: 29.86
RfactorNum. reflection% reflectionSelection details
Rfree0.339 177 4.61 %RANDOM
Rwork0.2975 ---
obs0.2995 3840 63.05 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refinement stepCycle: LAST / Resolution: 2.801→31.473 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1980 0 3 0 1983
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0042037
ELECTRON CRYSTALLOGRAPHYf_angle_d0.7762761
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d13.181408
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.047286
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.004359
LS refinement shellResolution: 2.801→2.97 Å
RfactorNum. reflection% reflection
Rfree0.339 177 -
Rwork0.2975 3663 -
obs--63 %
Refinement TLS params.

Method: refined / Refine-ID: ELECTRON CRYSTALLOGRAPHY

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.0278-0.00990.00170.1950.02430.17640.0165-0.0482-0.01250.0139-0.0540.05830.09320.0384-0.34860.00610.0250.0314-0.26180.097-0.0176-3.4312-9.392626.2232
20.0711-0.0021-0.03130.0642-0.01850.0478-0.03010.0513-0.0465-0.0787-0.025-0.1316-0.061-0.01-0.10190.02160.0256-0.05720.1761-0.11590.29038.1831-36.101812.324
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1ELECTRON CRYSTALLOGRAPHY1(chain A and resseq 1:129)
2ELECTRON CRYSTALLOGRAPHY2(chain B and resseq 1:129)

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