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Basic information

Entry
Database: PDB / ID: 5o4w
TitleProtein structure determination by electron diffraction using a single three-dimensional nanocrystal
ComponentsLysozyme C
KeywordsHYDROLASE / Lysozyme / Nanocrystal
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
Biological speciesGallus gallus (chicken)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.11 Å
AuthorsClabbers, M.T.B. / van Genderen, E. / Wan, W. / Wiegers, E.L. / Gruene, T. / Abrahams, J.P.
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2017
Title: Protein structure determination by electron diffraction using a single three-dimensional nanocrystal.
Authors: M T B Clabbers / E van Genderen / W Wan / E L Wiegers / T Gruene / J P Abrahams /
Abstract: Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of ...Three-dimensional nanometre-sized crystals of macromolecules currently resist structure elucidation by single-crystal X-ray crystallography. Here, a single nanocrystal with a diffracting volume of only 0.14 µm, i.e. no more than 6 × 10 unit cells, provided sufficient information to determine the structure of a rare dimeric polymorph of hen egg-white lysozyme by electron crystallography. This is at least an order of magnitude smaller than was previously possible. The molecular-replacement solution, based on a monomeric polyalanine model, provided sufficient phasing power to show side-chain density, and automated model building was used to reconstruct the side chains. Diffraction data were acquired using the rotation method with parallel beam diffraction on a Titan Krios transmission electron microscope equipped with a novel in-house-designed 1024 × 1024 pixel Timepix hybrid pixel detector for low-dose diffraction data collection. Favourable detector characteristics include the ability to accurately discriminate single high-energy electrons from X-rays and count them, fast readout to finely sample reciprocal space and a high dynamic range. This work, together with other recent milestones, suggests that electron crystallography can provide an attractive alternative in determining biological structures.
History
DepositionMay 31, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 23, 2017Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2017Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 17, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_ncs_dom_lim
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ncs_dom_lim.beg_auth_comp_id / _struct_ncs_dom_lim.beg_label_asym_id / _struct_ncs_dom_lim.beg_label_comp_id / _struct_ncs_dom_lim.beg_label_seq_id / _struct_ncs_dom_lim.end_auth_comp_id / _struct_ncs_dom_lim.end_label_asym_id / _struct_ncs_dom_lim.end_label_comp_id / _struct_ncs_dom_lim.end_label_seq_id

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Structure visualization

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Assembly

Deposited unit
A: Lysozyme C
B: Lysozyme C


Theoretical massNumber of molelcules
Total (without water)28,6622
Polymers28,6622
Non-polymers00
Water0
1
A: Lysozyme C


Theoretical massNumber of molelcules
Total (without water)14,3311
Polymers14,3311
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Lysozyme C


Theoretical massNumber of molelcules
Total (without water)14,3311
Polymers14,3311
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)104.560, 68.050, 32.050
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number18
Space group name H-MP21212
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B

NCS domain segments:

Component-ID: 1 / Ens-ID: 1 / Beg auth comp-ID: LYS / Beg label comp-ID: LYS / End auth comp-ID: LEU / End label comp-ID: LEU / Refine code: 1 / Auth seq-ID: 1 - 129 / Label seq-ID: 1 - 129

Dom-IDAuth asym-IDLabel asym-ID
1AA
2BB

NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2given(-0.949169, -0.314764, -0.001455), (-0.314767, 0.949162, 0.003602), (0.000247, 0.003876, -0.999992)31.45746, 24.94121, 80.54252

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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

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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 C / Type: COMPLEX / Entity ID: all / Source: NATURAL
Source (natural)Organism: Gallus gallus (chicken)
Buffer solutionpH: 3.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 0.01 e/Å2 / Film or detector model: OTHER
EM diffractionCamera length: 1890 mm
EM diffraction shellResolution: 41.44→2.11 Å / Fourier space coverage: 48.38 % / Multiplicity: 1.9 / Num. of structure factors: 6717 / Phase residual: 179.9 °
EM diffraction statsDetails: Parameters where extreme values were added are not applicable to our experiment and can not be filled out because there is no experimental data, these include overall phase error, overall ...Details: Parameters where extreme values were added are not applicable to our experiment and can not be filled out because there is no experimental data, these include overall phase error, overall phase residual and phase error rejection
Fourier space coverage: 48.38 % / High resolution: 2.11 Å / Num. of intensities measured: 12601 / Num. of structure factors: 6717 / Phase error: 179.9 ° / Phase residual: 179.9 ° / Phase error rejection criteria: 9999 / Rmerge: 26.3 / Rsym: 26.4

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 104.56 Å / B: 68.05 Å / C: 32.05 Å / Space group name: P21212 / Space group num: 18
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2ybl

2ybl


Resolution: 2.11→41.46 Å / Cor.coef. Fo:Fc: 0.848 / SU B: 10.205 / SU ML: 0.275 / Cross valid method: NONE
Details: We present Rcomplete instead of Rfree, calculating Rcomplete takes into account all reflections which is preferred when considering data sets with less than about 10,000 unique reflections ...Details: We present Rcomplete instead of Rfree, calculating Rcomplete takes into account all reflections which is preferred when considering data sets with less than about 10,000 unique reflections [Brunger (1997) Methods in Enzymology 277:366-396, Luebben & Gruene (2015) PNAS 112:8999-9003]. To calculate Rcomplete a 0.2% test set size was chosen i.e. 500 test sets were created. First all non-measured observations were removed from the reflection file, and the 500 test sets were assigned at random. The model was then refined independently each time omitting one of the different test sets, thus in total 500 iterations. Each time refinement was carried out until practically reaching convergence. Finally, the value for Rcomplete was calculated from the excluded data i.e. calculated from all reflections. Since all structure factors are used in turn this leads to a more robust calculation than Rfree.
RfactorNum. reflection% reflection
Rfree0.35 6717 100 %
Rwork0.335 --
obs-6717 48.38 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 23.986 Å2
Baniso -1Baniso -2Baniso -3
1--0.02 Å20 Å20 Å2
2---0.02 Å20 Å2
3---0.03 Å2
Refinement stepCycle: 1 / Total: 2000
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0170.0192045
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0060.021808
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.7841.9012768
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.20834170
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg6.5635256
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg37.05823100
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg19.25515332
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg16.4351522
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0980.2288
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0070.022336
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0040.02470
ELECTRON CRYSTALLOGRAPHYr_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_nbd_other
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined
ELECTRON CRYSTALLOGRAPHYr_nbtor_other
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_other
ELECTRON CRYSTALLOGRAPHYr_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_mcbond_it0.7492.4531030
ELECTRON CRYSTALLOGRAPHYr_mcbond_other0.7482.4531029
ELECTRON CRYSTALLOGRAPHYr_mcangle_it1.0083.6791284
ELECTRON CRYSTALLOGRAPHYr_mcangle_other1.0083.6791285
ELECTRON CRYSTALLOGRAPHYr_scbond_it0.7482.4911013
ELECTRON CRYSTALLOGRAPHYr_scbond_other0.7472.4911014
ELECTRON CRYSTALLOGRAPHYr_scangle_it
ELECTRON CRYSTALLOGRAPHYr_scangle_other1.013.7181484
ELECTRON CRYSTALLOGRAPHYr_long_range_B_refined1.56346.2688241
ELECTRON CRYSTALLOGRAPHYr_long_range_B_other1.56346.2658241
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr
ELECTRON CRYSTALLOGRAPHYr_sphericity_free
ELECTRON CRYSTALLOGRAPHYr_sphericity_bonded
Refine LS restraints NCSNumber: 1000 / Type: tight thermal / Rms dev position: 0.84 Å / Weight position: 0.5
LS refinement shellResolution: 2.11→2.16 Å
RfactorNum. reflection% reflection
Rwork0.323 504 -
obs--49.95 %

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