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- PDB-7uly: MicroED structure of triclinic lysozyme -

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Basic information

Entry
Database: PDB / ID: 7uly
TitleMicroED structure of triclinic lysozyme
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
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
NITRATE ION / Lysozyme C
Similarity search - Component
Biological speciesGallus gallus (chicken)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 0.87 Å
AuthorsClabbers, M.T.B. / Martynowycz, M.W. / 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)GM136508 United States
CitationJournal: J Struct Biol X / Year: 2022
Title: Hydrogens and hydrogen-bond networks in macromolecular MicroED data.
Authors: Max T B Clabbers / Michael W Martynowycz / Johan Hattne / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray ...Microcrystal electron diffraction (MicroED) is a powerful technique utilizing electron cryo-microscopy (cryo-EM) for protein structure determination of crystalline samples too small for X-ray crystallography. Electrons interact with the electrostatic potential of the sample, which means that the scattered electrons carry information about the charged state of atoms and provide relatively stronger contrast for visualizing hydrogen atoms. Accurately identifying the positions of hydrogen atoms, and by extension the hydrogen bonding networks, is of importance for understanding protein structure and function, in particular for drug discovery. However, identification of individual hydrogen atom positions typically requires atomic resolution data, and has thus far remained elusive for macromolecular MicroED. Recently, we presented the structure of triclinic hen egg-white lysozyme at 0.87 Å resolution. The corresponding data were recorded under low exposure conditions using an electron-counting detector from thin crystalline lamellae. Here, using these subatomic resolution MicroED data, we identified over a third of all hydrogen atom positions based on strong difference peaks, and directly visualize hydrogen bonding interactions and the charged states of residues. Furthermore, we find that the hydrogen bond lengths are more accurately described by the inter-nuclei distances than the centers of mass of the corresponding electron clouds. We anticipate that MicroED, coupled with ongoing advances in data collection and refinement, can open further avenues for structural biology by uncovering the hydrogen atoms and hydrogen bonding interactions underlying protein structure and function.
History
DepositionApr 5, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 15, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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
Water2,018112
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area630 Å2
ΔGint4 kcal/mol
Surface area6150 Å2
MethodPISA
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.: 112 / 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 weightValue: 0.0144 MDa / Experimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Buffer solutionpH: 4.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Protein crystal lamellae
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
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1536 mm
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
IDNameVersionCategory
8REFMAC5.8.0267model refinement
12AIMLESScrystallography merging
13REFMAC5.8.02673D 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: 11.981 / 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: 0.949 / SU B: 1.286 / SU ML: 0.031 / Cross valid method: THROUGHOUT / ESU R: 0.028 / ESU R Free: 0.028
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2197 3168 4.876 %
Rwork0.1973 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: 11.981 Å2
Baniso -1Baniso -2Baniso -3
1--1.18 Å2-0.119 Å2-0.194 Å2
2--0.174 Å20.142 Å2
3---0.861 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0340.0131138
ELECTRON CRYSTALLOGRAPHYr_bond_other_d00.0141042
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg2.4471.6371550
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg2.0761.62390
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg7.0935148
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg32.0820.29468
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg14.23115188
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg17.1751513
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.1690.2141
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0120.021378
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0050.02316
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2480.2404
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_other0.2290.21515
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.2290.2694
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbtor_other0.1080.2877
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.1330.2137
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.2780.228
ELECTRON CRYSTALLOGRAPHYr_nbd_other0.1980.290
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_refined0.1290.239
ELECTRON CRYSTALLOGRAPHYr_mcbond_it1.3421.017558
ELECTRON CRYSTALLOGRAPHYr_mcbond_other1.3346.514557
ELECTRON CRYSTALLOGRAPHYr_mcangle_it1.5771.522708
ELECTRON CRYSTALLOGRAPHYr_mcangle_other1.5764.974709
ELECTRON CRYSTALLOGRAPHYr_scbond_it5.3381.292580
ELECTRON CRYSTALLOGRAPHYr_scbond_other5.2914104.889568
ELECTRON CRYSTALLOGRAPHYr_scangle_it3.611.852838
ELECTRON CRYSTALLOGRAPHYr_scangle_other3.5681.835826
ELECTRON CRYSTALLOGRAPHYr_lrange_it3.42821.9826885
ELECTRON CRYSTALLOGRAPHYr_lrange_other3.3221.7436776
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr12.02531138
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
0.87-0.8930.385940.38818540.38854930.0120.01635.46330.39
0.893-0.9170.4761660.4631450.46153670.0240.03361.69180.476
0.917-0.9430.4511850.42937300.4352010.6680.62975.2740.436
0.943-0.9720.3931930.37538500.37650040.7670.79980.79540.37
0.972-1.0040.3622460.34141200.34348940.8610.86689.21130.326
1.004-1.0390.3142080.28641180.28747140.9230.91191.76920.266
1.039-1.0790.2442120.2241710.22145530.9410.94496.26620.199
1.079-1.1220.2092270.17640840.17843970.9450.95798.04410.156
1.122-1.1720.1991890.15639400.15842130.9530.96298.00620.134
1.172-1.2290.1961740.15438620.15640460.9540.95999.75280.132
1.229-1.2950.181920.14636240.14738160.9560.9611000.125
1.295-1.3740.1851780.15634460.15836240.9510.9591000.142
1.374-1.4680.1831760.1632680.16234460.9530.96299.9420.152
1.468-1.5850.2061420.18129960.18231400.9250.94299.93630.172
1.585-1.7350.2061500.17527770.17629340.9490.9599.76140.166
1.735-1.9380.21100.17725220.17826450.920.93599.50850.169
1.938-2.2340.2071000.17522040.17623240.8910.92799.13940.161
2.234-2.7280.22940.17118590.17319730.9190.93698.98630.17
2.728-3.8220.199890.21314310.21315350.9350.92999.02280.208
3.822-19.8760.293430.3138050.3128640.9220.91398.14810.307

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