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- PDB-8e54: MicroED structure of triclinic lysozyme recorded on K3 -

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Basic information

Entry
Database: PDB / ID: 8.0E+54
TitleMicroED structure of triclinic lysozyme recorded on K3
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: 1.2 Å
AuthorsClabbers, M.T.B. / Martynowycz, M.W. / Hattne, J. / Nannenga, B.L. / 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 / Year: 2022
Title: Electron-counting MicroED data with the K2 and K3 direct electron detectors.
Authors: Max T B Clabbers / Michael W Martynowycz / Johan Hattne / Brent L Nannenga / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances ...Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.
History
DepositionAug 19, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 19, 2022Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AAA: Lysozyme C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)16,3202
Polymers16,2581
Non-polymers621
Water2,126118
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)26.380, 30.760, 33.000
Angle α, β, γ (deg.)87.851, 108.849, 112.600
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: 16257.660 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.: 1 / Source method: obtained synthetically / Formula: NO3
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 118 / 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: 0.1 sec. / Electron dose: 0.001 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of diffraction images: 5600 / Num. of grids imaged: 1 / Num. of real images: 1
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092
EM diffractionCamera length: 373 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
11AIMLESScrystallography merging
12REFMAC5.8.02673D reconstruction
13REFMAC5.8.0267model refinement
EM 3D crystal entity∠α: 87.85 ° / ∠β: 108.85 ° / ∠γ: 112.6 ° / A: 26.38 Å / B: 30.76 Å / C: 33 Å / Space group name: P-1 / Space group num: 2
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 12.94 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Maximum likelihood
RefinementResolution: 1.2→31.076 Å / Cor.coef. Fo:Fc: 0.97 / Cor.coef. Fo:Fc free: 0.944 / SU B: 3.756 / SU ML: 0.069 / Cross valid method: THROUGHOUT / ESU R: 0.068 / ESU R Free: 0.068
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.242 1186 4.838 %
Rwork0.1813 23326 -
all0.184 --
obs-24512 86.839 %
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: 12.937 Å2
Baniso -1Baniso -2Baniso -3
1-0.599 Å20.678 Å2-0.569 Å2
2--1.349 Å20.58 Å2
3----2.225 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0210.0131028
ELECTRON CRYSTALLOGRAPHYr_bond_other_d00.014938
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg2.1421.6341392
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.9291.5952136
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg7.3125128
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg32.98520.65661
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg13.02515166
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg16.5621511
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0920.2130
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0120.021214
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0020.02280
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2420.2386
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_other0.2220.21258
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.2120.2587
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbtor_other0.0940.2643
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.2850.2147
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.2470.230
ELECTRON CRYSTALLOGRAPHYr_nbd_other0.2210.284
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_refined0.150.225
ELECTRON CRYSTALLOGRAPHYr_mcbond_it1.8091.107515
ELECTRON CRYSTALLOGRAPHYr_mcbond_other1.7291.102514
ELECTRON CRYSTALLOGRAPHYr_mcangle_it2.0861.669642
ELECTRON CRYSTALLOGRAPHYr_mcangle_other2.1091.676643
ELECTRON CRYSTALLOGRAPHYr_scbond_it2.2031.406513
ELECTRON CRYSTALLOGRAPHYr_scbond_other2.1451.403510
ELECTRON CRYSTALLOGRAPHYr_scangle_it2.5282.004750
ELECTRON CRYSTALLOGRAPHYr_scangle_other2.4491.999747
ELECTRON CRYSTALLOGRAPHYr_lrange_it2.91422.6735206
ELECTRON CRYSTALLOGRAPHYr_lrange_other2.6322.1645090
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr4.78231966
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
1.2-1.2310.355760.32116820.32320900.7720.79684.11480.319
1.231-1.2650.328750.29316780.29420260.7860.83186.52520.29
1.265-1.3010.346930.28416240.28719870.80.8586.41170.281
1.301-1.3410.29850.26516260.26619360.8070.86488.37810.258
1.341-1.3850.352800.27515660.27918600.7890.84888.49460.269
1.385-1.4340.339680.27314970.27517720.7650.80888.31830.266
1.434-1.4880.298820.24714500.2517350.7950.83488.29970.239
1.488-1.5480.269710.20714270.2116870.8760.90988.79670.195
1.548-1.6170.242710.18413330.18715940.9070.93388.08030.171
1.617-1.6960.229630.1813060.18215440.9170.92888.66580.166
1.696-1.7870.255660.18811980.19214400.9090.92187.77780.175
1.787-1.8950.258640.17611620.18113910.9240.92488.1380.166
1.895-2.0250.206440.14410870.14613070.9430.95386.5340.136
2.025-2.1870.257570.149890.14612120.9310.95686.30360.138
2.187-2.3940.227410.1448830.14810980.950.95484.1530.145
2.394-2.6750.174410.1428090.14410010.9480.95984.91510.143
2.675-3.0850.205420.1437070.1478830.9460.95984.82450.151
3.085-3.7690.156360.1386020.1397550.960.96384.50330.155
3.769-5.2920.213170.1324800.1365870.9510.96184.66780.15
5.292-31.0760.262140.1962190.23210.8580.90872.58570.221

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