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- PDB-6law: MicroED structure of proteinase K at 1.50A determained using crys... -

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Basic information

Entry
Database: PDB / ID: 6law
TitleMicroED structure of proteinase K at 1.50A determained using crystal lamellas prepared by focused ion beam milling
ComponentsProteinase K
KeywordsHYDROLASE / Proteinase K / MicroED / Focused Ion Beam / crystal lamella
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / metal ion binding
Peptidase S8/S53 domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase S8, subtilisin-related / Peptidase S8, subtilisin, His-active site / Peptidase S8, subtilisin, Asp-active site / Peptidase S8, subtilisin, Ser-active site / Proteinase K-like catalytic domain / Peptidase S8/S53 domain superfamily / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Subtilase family
Proteinase K
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.5 Å
AuthorsZhou, H. / Luo, Z. / Li, X.
Funding support China, 4items
OrganizationGrant numberCountry
National Natural Science Foundation of China31570730 China
National Natural Science Foundation of China31722015 China
Ministry of Science and Technology (China)2016YFA0501102 China
Ministry of Science and Technology (China)2016YFA0501902 China
CitationJournal: J. Struct. Biol. / Year: 2019
Title: Using focus ion beam to prepare crystal lamella for electron diffraction.
Authors: Heng Zhou / Zhipu Luo / Xueming Li /
Abstract: Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the ...Electron diffraction provides a powerful tool to solve the structures of small protein crystals. However, strong interactions between the electrons and the materials limit the application of the electron crystallographic method on large protein crystals with micrometer or larger sizes. Here, we used the focused ion beam (FIB) equipped on the scanning electron microscope (SEM) to mill a large crystal to thin lamella. The influences of the milling on the crystal lamella were observed and investigated, including radiation damage on the crystal surface during the FIB imaging, deformation of the thin crystal lamella, and variation in the diffraction intensities under electron radiation. These observations provide important information to optimize the FIB milling, and hence is important to obtain high-quality crystal samples for routine structure determination of protein crystals using the electron cryo-microscope.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 13, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 4, 2019Provider: repository / Type: Initial release

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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0552
Polymers28,9591
Non-polymers961
Water4,143230
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area180 Å2
ΔGint-13 kcal/mol
Surface area10810 Å2
Unit cell
γ
α
β
Length a, b, c (Å)69.314, 69.314, 104.116
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Components on special symmetry positions
IDModelComponents
11A-479-

HOH

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Components

#1: Protein/peptide Proteinase K / / Endopeptidase K / Tritirachium alkaline proteinase


Mass: 28958.791 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4 / Sulfate
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 230 / Source method: isolated from a natural source / Formula: H2O / Water
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: proteinase K / Type: COMPLEX / Entity ID: 1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Parengyodontium album (fungus)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 0.028 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
EM diffractionCamera length: 1000 mm
EM diffraction shellResolution: 1.5→1.55 Å / Fourier space coverage: 90.6 % / Multiplicity: 5.2 / Num. of structure factors: 3693 / Phase residual: 1 °
EM diffraction statsFourier space coverage: 91.1 % / High resolution: 1.5 Å / Num. of intensities measured: 200471 / Num. of structure factors: 37742 / Phase error: 0 ° / Phase residual: 1 ° / Phase error rejection criteria: 60 / Rmerge: 0.245 / Rsym: 0.245
DetectorDate: Apr 21, 2018

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0222refinement
PDB_EXTRACT3.25data extraction
EM 3D crystal entityAngle alpha: 90 ° / Angle beta: 90 ° / Angle gamma: 90 ° / Length a: 69.31 Å / Length b: 69.31 Å / Length c: 104.12 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 1.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: RECIPROCAL
RefinementResolution: 1.5→12.31 Å / Cor.coef. Fo:Fc: 0.956 / Cor.coef. Fo:Fc free: 0.938 / SU B: 2.195 / SU ML: 0.077 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.087 / ESU R Free: 0.09 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2251 1893 5 %RANDOM
Rwork0.1868 ---
Obs0.1888 35768 91.07 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: BABINET MODEL WITH MASK
Displacement parametersBiso max: 71.5 Å2 / Biso mean: 10.998 Å2 / Biso min: 3.37 Å2
Baniso -1Baniso -2Baniso -3
1--0.1 Å20 Å20 Å2
2---0.1 Å20 Å2
3---0.2 Å2
Refinement stepCycle: final / Resolution: 1.5→12.31 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2032 0 5 230 2267
Biso mean--13.15 22.02 -
Num. residues----279
LS refinement shellResolution: 1.5→1.538 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.311 142 -
Rwork0.315 2549 -
All-2691 -
Obs--90.51 %

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