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- PDB-8fyo: MicroED structure of Proteinase K from lamellae milled from multi... -

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Basic information

Entry
Database: PDB / ID: 8fyo
TitleMicroED structure of Proteinase K from lamellae milled from multiple plasma sources
ComponentsProteinase K
KeywordsHYDROLASE
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding
Similarity search - Function
Proteinase K-like catalytic domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site ...Proteinase K-like catalytic domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Serine proteases, subtilase domain profile. / Peptidase S8, subtilisin-related / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain
Similarity search - Domain/homology
NITRATE ION / Proteinase K
Similarity search - Component
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.39 Å
AuthorsMartynowycz, M.W. / Shiriaeva, A. / Clabbers, M.T.B. / Nicolas, W.J. / Weaver, S.J. / 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 Commun / Year: 2023
Title: A robust approach for MicroED sample preparation of lipidic cubic phase embedded membrane protein crystals.
Authors: Michael W Martynowycz / Anna Shiriaeva / Max T B Clabbers / William J Nicolas / Sara J Weaver / Johan Hattne / Tamir Gonen /
Abstract: Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction ...Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.
History
DepositionJan 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 24, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,1014
Polymers28,9591
Non-polymers1423
Water5,116284
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)67.230, 67.230, 107.860
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Space group name HallP4nw2abw
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2
Components on special symmetry positions
IDModelComponents
11A-595-

HOH

21A-740-

HOH

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Components

#1: Protein 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-NO3 / NITRATE ION / Nitrate


Mass: 62.005 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: NO3
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 284 / 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: Proteinase K / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.0289 MDa / Experimental value: NO
Source (natural)Organism: Parengyodontium album (fungus)
EM crystal formationLipid mixture: None
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Milled microcrystals
Specimen supportGrid 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 / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / Calibrated defocus max: 0 nm / 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: 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
ReflectionBiso Wilson estimate: 11.45 Å2

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Processing

Software
NameVersionClassification
phenix.refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM softwareName: AIMLESS / Category: crystallography merging
Image processingDetails: Binned by 2
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.02 Å / B: 67.02 Å / C: 107.53 Å / Space group name: 96 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 1.39 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 11.7 / Protocol: AB INITIO MODEL / Space: RECIPROCAL / Target criteria: Maximum likelihood
RefinementResolution: 1.39→19.83 Å / SU ML: 0.1451 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 14.5854
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.163 2500 4.98 %
Rwork0.1252 47661 -
obs0.1271 50161 99.42 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 13.61 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.01362134
ELECTRON CRYSTALLOGRAPHYf_angle_d1.15572914
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0909324
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0116387
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d5.7368326
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.39-1.420.31551200.28242491ELECTRON CRYSTALLOGRAPHY95.08
1.42-1.450.26051360.23482604ELECTRON CRYSTALLOGRAPHY98.6
1.45-1.480.28021340.21452592ELECTRON CRYSTALLOGRAPHY98.91
1.48-1.510.25061360.19312594ELECTRON CRYSTALLOGRAPHY99.71
1.51-1.550.22921600.17592610ELECTRON CRYSTALLOGRAPHY99.86
1.55-1.590.21771510.1582595ELECTRON CRYSTALLOGRAPHY99.82
1.59-1.640.22861390.15582656ELECTRON CRYSTALLOGRAPHY99.89
1.64-1.690.21641260.14042624ELECTRON CRYSTALLOGRAPHY99.85
1.69-1.750.18141510.13092600ELECTRON CRYSTALLOGRAPHY99.93
1.75-1.820.16721220.12252670ELECTRON CRYSTALLOGRAPHY99.89
1.82-1.90.16271280.10282659ELECTRON CRYSTALLOGRAPHY99.82
1.9-20.14591360.09782647ELECTRON CRYSTALLOGRAPHY99.86
2-2.130.15771340.10232659ELECTRON CRYSTALLOGRAPHY99.93
2.13-2.290.15411330.10032674ELECTRON CRYSTALLOGRAPHY99.89
2.29-2.520.1281480.10742682ELECTRON CRYSTALLOGRAPHY99.89
2.52-2.890.14491530.11062695ELECTRON CRYSTALLOGRAPHY99.82
2.89-3.640.13191250.11242754ELECTRON CRYSTALLOGRAPHY99.79
3.64-19.830.12861680.11272855ELECTRON CRYSTALLOGRAPHY98.98

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