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- PDB-6pq4: LCP-embedded Proteinase K treated with lipase -

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Basic information

Entry
Database: PDB / ID: 6pq4
TitleLCP-embedded Proteinase K treated with lipase
ComponentsProteinase K
KeywordsHYDROLASE / Proteinase K / LCP / lipase / MicroED
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / proteolysis / extracellular region / 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 / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2 Å
AuthorsBu, G. / Zhu, L. / Jing, L. / Shi, D. / Gonen, T. / Liu, W. / Nannenga, B.L.
Funding support United States, 4items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)NSF MRI 1531991 United States
National Science Foundation (NSF, United States)1231306 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)R21DA042298 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM124152 United States
CitationJournal: Structure / Year: 2020
Title: Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED.
Authors: Lan Zhu / Guanhong Bu / Liang Jing / Dan Shi / Ming-Yue Lee / Tamir Gonen / Wei Liu / Brent L Nannenga /
Abstract: The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization ...The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy to collect electron diffraction data and determine high-resolution structures from very thin micro- and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.
History
DepositionJul 8, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 5, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 12, 2020Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Oct 21, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0734
Polymers28,9311
Non-polymers1423
Water1,71195
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area300 Å2
ΔGint-19 kcal/mol
Surface area10340 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.561, 67.561, 106.802
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-509-

HOH

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Components

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


Mass: 28930.783 Da / Num. of mol.: 1 / Fragment: UNP residues 106-384 / Source method: isolated from a natural source / Source: (natural) Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NO3 / NITRATE ION / Nitrate


Mass: 62.005 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: NO3
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 95 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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
Source (natural)Organism: Parengyodontium album (fungus)
Buffer solutionpH: 6.5
SpecimenConc.: 40 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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: OTHER
Electron lensMode: DIFFRACTION
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 4 sec. / Electron dose: 0.04 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k)
EM diffractionCamera length: 1940 mm
EM diffraction shellResolution: 2.05→2 Å / Fourier space coverage: 94.8 % / Multiplicity: 5.4 / Num. of structure factors: 1191 / Phase residual: 1.0E-5 °
EM diffraction statsFourier space coverage: 94.6 % / High resolution: 2 Å / Num. of intensities measured: 85421 / Num. of structure factors: 16351 / Phase error: 0 ° / Phase residual: 1.0E-5 ° / Phase error rejection criteria: 0 / Rmerge: 40.4 / Rsym: 40.4

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Processing

Software
NameClassification
PHENIXrefinement
PHASERphasing
iMOSFLMdata reduction
SCALAdata scaling
EM software
IDNameVersionCategoryDetails
8PHENIX1.13-2998molecular replacementPhaser using search model ID 2ID8
11CCP4 package7.0.058crystallography merging
12PHENIX1.13-29983D reconstruction
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.56 Å / B: 67.56 Å / C: 106.8 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 2 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 2ID8

2id8


Resolution: 2→16.58 Å / Cross valid method: FREE R-VALUE
RfactorNum. reflection% reflection
Rfree0.2824 1632 10 %
Rwork0.2442 --
obs-16351 94.6 %
LS refinement shellResolution: 2.0001→2.0589 Å
RfactorNum. reflection% reflection
Rfree0.3666 133 -
Rwork0.3356 1200 -
obs--94.6 %

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