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- PDB-7skw: Ab initio structure of triclinic lysozyme from electron-counted M... -

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Basic information

Entry
Database: PDB / ID: 7skw
TitleAb initio structure of triclinic lysozyme from electron-counted MicroED data
ComponentsLysozyme C
KeywordsHYDROLASE
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
NITRATE ION / Lysozyme C
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 0.87 Å
AuthorsMartynowycz, M.W. / Clabbers, M.T.B. / Hattne, J. / Gonen, T.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
CitationJournal: Nat Methods / Year: 2022
Title: Ab initio phasing macromolecular structures using electron-counted MicroED data.
Authors: Michael W Martynowycz / Max T B Clabbers / Johan Hattne / Tamir Gonen /
Abstract: Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals ...Structures of two globular proteins were determined ab initio using microcrystal electron diffraction (MicroED) data that were collected on a direct electron detector in counting mode. Microcrystals were identified using a scanning electron microscope (SEM) and thinned with a focused ion beam (FIB) to produce crystalline lamellae of ideal thickness. Continuous-rotation data were collected using an ultra-low exposure rate to enable electron counting in diffraction. For the first sample, triclinic lysozyme extending to a resolution of 0.87 Å, an ideal helical fragment of only three alanine residues provided initial phases. These phases were improved using density modification, allowing the entire atomic structure to be built automatically. A similar approach was successful on a second macromolecular sample, proteinase K, which is much larger and diffracted to a resolution of 1.5 Å. These results demonstrate that macromolecules can be determined to sub-ångström resolution by MicroED and that ab initio phasing can be successfully applied to counting data.
History
DepositionOct 21, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 8, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 15, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Jun 22, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 9, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Lysozyme C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,5795
Polymers14,3311
Non-polymers2484
Water2,810156
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)26.420, 30.720, 33.010
Angle α, β, γ (deg.)88.319, 109.095, 112.075
Int Tables number1
Space group name H-MP1

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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-NO3 / NITRATE ION


Mass: 62.005 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: NO3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 156 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

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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 weightValue: 0.0144 MDa / Experimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Buffer solutionpH: 4.7
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Milled microcrystals
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Data collection

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingAverage exposure time: 0.5 sec. / Electron dose: 0.001 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 840 / Num. of grids imaged: 1 / Num. of real images: 1
Details: 0.15 degrees per second, 0.5 second readout, 30 to -30 degrees
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1202 mm / Tilt angle list: -30,30
EM diffraction shellResolution: 0.87→0.9 Å / Fourier space coverage: 37.64 % / Multiplicity: 2.1 / Num. of structure factors: 2783 / Phase residual: 30 °
EM diffraction statsFourier space coverage: 87.58 % / High resolution: 0.87 Å / Num. of intensities measured: 569407 / Num. of structure factors: 64974 / Phase error: 30 ° / Phase error rejection criteria: None / Rmerge: 0.236 / Rsym: 0.073

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Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement / Contact author: Garib N. Murshudov / Contact author email: garib[at]mrc-lmb.cam.ac.uk / Date: 2020-24-08
Description: (un)restrained refinement or idealisation of macromolecular structures
EM software
IDNameCategory
8REFMACmodel refinement
12AIMLESScrystallography merging
13REFMAC3D reconstruction
Image processingDetails: Binned by 2
EM 3D crystal entity∠α: 88.319 ° / ∠β: 109.095 ° / ∠γ: 112.075 ° / A: 26.42 Å / B: 30.72 Å / C: 33.01 Å / Space group name: P1 / Space group num: 1
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 12.188 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Maximum likelihood
RefinementResolution: 0.87→19.876 Å / Cor.coef. Fo:Fc: 0.951 / Cor.coef. Fo:Fc free: 0.951 / SU B: 1.263 / SU ML: 0.03 / Cross valid method: FREE R-VALUE / ESU R: 0.028 / ESU R Free: 0.028
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflectionSelection details
Rfree0.2214 3168 4.876 %Random selection
Rwork0.1969 61806 --
all0.198 ---
obs-64974 87.578 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 12.188 Å2
Baniso -1Baniso -2Baniso -3
1--0.65 Å2-0.32 Å2-0.122 Å2
2--0.143 Å2-0.056 Å2
3---0.652 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0330.0131050
ELECTRON CRYSTALLOGRAPHYr_bond_other_d00.018955
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg2.2881.6351420
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg2.0781.5982180
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg7.0115131
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg31.15420.80662
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg12.11415171
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg17.9781511
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.1530.2132
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0120.021243
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0020.02283
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2420.2357
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_other0.220.21343
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.2250.2630
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbtor_other0.1070.2775
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.1360.2142
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_other0.050.21
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.2010.243
ELECTRON CRYSTALLOGRAPHYr_nbd_other0.2310.293
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_refined0.320.256
ELECTRON CRYSTALLOGRAPHYr_mcbond_it1.330.924519
ELECTRON CRYSTALLOGRAPHYr_mcbond_other1.2910.918518
ELECTRON CRYSTALLOGRAPHYr_mcangle_it1.5841.39647
ELECTRON CRYSTALLOGRAPHYr_mcangle_other1.5841.397648
ELECTRON CRYSTALLOGRAPHYr_scbond_it1.9281.187531
ELECTRON CRYSTALLOGRAPHYr_scbond_other1.8031.178519
ELECTRON CRYSTALLOGRAPHYr_scangle_it2.3771.699771
ELECTRON CRYSTALLOGRAPHYr_scangle_other2.2081.686759
ELECTRON CRYSTALLOGRAPHYr_lrange_it5.78620.5485494
ELECTRON CRYSTALLOGRAPHYr_lrange_other2.5218.8025325
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr3.00132005
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
0.87-0.8930.41940.39718540.39754930.0510.06335.46330.399
0.893-0.9170.4621660.46331450.46353670.0160.02761.69180.475
0.917-0.9430.4481850.43637300.43652010.0360.02775.2740.44
0.943-0.9720.3991930.38838500.38850040.2510.26880.79540.38
0.972-1.0040.3732460.35241200.35348940.8090.81389.21130.338
1.004-1.0390.3122080.30441180.30447140.6470.64991.76920.286
1.039-1.0790.2432120.21841710.21945530.9420.93796.26620.199
1.079-1.1220.22270.17640840.17743970.9440.95498.04410.157
1.122-1.1720.2011890.15539400.15742130.9490.95698.00620.134
1.172-1.2290.191740.15338620.15440460.9510.95599.75280.131
1.229-1.2950.1721920.14136240.14338160.9550.961000.123
1.295-1.3740.1851780.15434460.15536240.950.9581000.141
1.374-1.4680.1861760.1632680.16234460.950.9699.9420.153
1.468-1.5850.2161420.17829960.1831400.9270.94699.93630.172
1.585-1.7350.1971500.17727770.17829340.9490.94999.76140.17
1.735-1.9380.2111100.17725220.17926450.9190.93799.50850.169
1.938-2.2340.2241000.17522040.17723240.9010.93199.13940.162
2.234-2.7280.2940.16418590.16619730.9350.94598.98630.161
2.728-3.8220.207890.21314310.21315350.9370.93199.02280.208
3.822-19.8760.304430.3168050.3158640.9260.9198.14810.31

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