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- PDB-6pq0: LCP-embedded Proteinase K treated with MPD -

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Basic information

Entry
Database: PDB / ID: 6pq0
TitleLCP-embedded Proteinase K treated with MPD
ComponentsProteinase K
KeywordsHYDROLASE / Proteinase K / LCP / MPD / microED
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
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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Structure visualization

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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0113
Polymers28,9311
Non-polymers802
Water1,60389
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area170 Å2
ΔGint-20 kcal/mol
Surface area10210 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.340, 67.340, 106.475
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212
Components on special symmetry positions
IDModelComponents
11A-507-

HOH

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Components

#1: Protein Proteinase K


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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 89 / 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→2.05 Å / Fourier space coverage: 63.3 % / Multiplicity: 6.3 / Num. of structure factors: 782 / Phase residual: 1.0E-6 °
EM diffraction statsFourier space coverage: 84.6 % / High resolution: 2 Å / Num. of intensities measured: 111081 / Num. of structure factors: 14491 / Phase error: 0 ° / Phase residual: 1.0E-6 ° / Phase error rejection criteria: 0 / Rmerge: 32.4 / Rsym: 32.4
ReflectionHighest resolution: 2.0002 Å
Reflection shellResolution: 2.0002→2.0716 Å / Redundancy: 6.3 % / Mean I/σ(I) obs: 3.4 / Num. unique obs: 782 / CC1/2: 0.368 / % possible all: 63

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Processing

Software
NameVersionClassification
PHENIX(1.13_2998: ???)refinement
PHASERphasing
iMOSFLMdata reduction
SCALAdata scaling
EM software
IDNameVersionCategoryDetails
9PHENIX1.13-2998molecular replacementPhaser using search model ID 2ID8
12CCP4 package7.0.058crystallography merging
13PHENIX1.13-29983D reconstruction
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.34 Å / B: 67.34 Å / C: 106.47 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→17.39 Å / SU ML: 0.24 / Cross valid method: FREE R-VALUE / σ(F): 1.4 / Phase error: 21.59
RfactorNum. reflection% reflection
Rfree0.2665 1444 10.01 %
Rwork0.2168 --
obs0.2218 14432 84.12 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0032070
ELECTRON CRYSTALLOGRAPHYf_angle_d0.5712814
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d2.8231208
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.044312
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.003370
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.0002-2.07160.33021050.2682953ELECTRON CRYSTALLOGRAPHY63
2.0716-2.15440.34911250.27391113ELECTRON CRYSTALLOGRAPHY74
2.1544-2.25230.32921360.26291231ELECTRON CRYSTALLOGRAPHY81
2.2523-2.37070.33861370.26341227ELECTRON CRYSTALLOGRAPHY81
2.3707-2.51890.28171400.24961258ELECTRON CRYSTALLOGRAPHY83
2.5189-2.71270.31191570.23481418ELECTRON CRYSTALLOGRAPHY92
2.7127-2.98440.27381560.21821406ELECTRON CRYSTALLOGRAPHY92
2.9844-3.41340.26371590.19891426ELECTRON CRYSTALLOGRAPHY92
3.4134-4.28990.18211600.16251442ELECTRON CRYSTALLOGRAPHY92
4.2899-17.38780.18821690.16291514ELECTRON CRYSTALLOGRAPHY90

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