[English] 日本語
Yorodumi
- PDB-8sdk: The MicroED structure of proteinase K crystallized by suspended d... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8sdk
TitleThe MicroED structure of proteinase K crystallized by suspended drop crystallization
ComponentsProteinase K
KeywordsHYDROLASE / MicroED / suspended drop crystallization / enzyme
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. ...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 / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Peptidase S8/S53 domain / Subtilase family
Similarity search - Domain/homology
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2.1 Å
AuthorsGillman, C. / Nicolas, W.J. / Martynowycz, M.W. / Gonen, T.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
Department of Defense (DOD, United States)HDTRA1-21-1-0004 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: IUCrJ / Year: 2023
Title: Design and implementation of suspended drop crystallization.
Authors: Cody Gillman / William J Nicolas / Michael W Martynowycz / Tamir Gonen /
Abstract: In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an ...In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.
History
DepositionApr 6, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 31, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 14, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 12, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0673
Polymers28,9311
Non-polymers1362
Water1,946108
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)68.260, 68.260, 101.950
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

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


Mass: 28930.783 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
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 108 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationY

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

-
Sample preparation

ComponentName: Proteinase K from Tritirachium album / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.0289 MDa / Experimental value: NO
Source (natural)Organism: Parengyodontium album (fungus)
Buffer solutionpH: 8
SpecimenConc.: 25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

-
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
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 0.64 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 700 / Num. of real images: 700
Details: Detector distance: 1550 mm Images collected 60 degrees from -30 to +30 tilt
EM diffractionCamera length: 1550 mm / Tilt angle list: -30,30
EM diffraction shellResolution: 2.1→30.42 Å / Fourier space coverage: 87 % / Multiplicity: 4.41 / Num. of structure factors: 12774 / Phase residual: 31 °
EM diffraction statsFourier space coverage: 87 % / High resolution: 2.1 Å / Num. of intensities measured: 56301 / Num. of structure factors: 12774 / Phase error rejection criteria: none / Rmerge: 44.5
ReflectionBiso Wilson estimate: 16.75 Å2

-
Processing

SoftwareName: PHENIX / Version: 1.20.1_4487 / Classification: refinement / Contact author: Paul D. Adams / Contact author email: pdadams[at]lbl.gov / Language: Python/C++ / URL: https://www.phenix-online.org/ / Type: program
EM softwareName: PHENIX / Category: model refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 68.26 Å / B: 68.26 Å / C: 101.95 Å / Space group name: P41212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 2.1 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
RefinementResolution: 2.1→30.42 Å / SU ML: 0.3807 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 31.4508
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Details: None
RfactorNum. reflection% reflection
Rfree0.2941 2208 10 %
Rwork0.2547 19866 -
obs0.2587 22074 82.15 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 16.09 Å2
Refinement stepCycle: LAST / Resolution: 2.1→30.42 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2029 0 6 108 2143
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.00142072
ELECTRON CRYSTALLOGRAPHYf_angle_d0.38292816
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.0393312
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.0022370
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d3.6066307
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.1-2.150.41871380.36021206ELECTRON CRYSTALLOGRAPHY79.34
2.15-2.20.36621280.34891225ELECTRON CRYSTALLOGRAPHY80.54
2.2-2.250.3921290.34221194ELECTRON CRYSTALLOGRAPHY79.36
2.25-2.310.39971420.34611212ELECTRON CRYSTALLOGRAPHY80.17
2.31-2.380.31811350.31681223ELECTRON CRYSTALLOGRAPHY81.51
2.38-2.460.32051350.31611238ELECTRON CRYSTALLOGRAPHY81.39
2.46-2.540.37381390.31841232ELECTRON CRYSTALLOGRAPHY81.32
2.54-2.650.36571360.31041240ELECTRON CRYSTALLOGRAPHY82.89
2.65-2.770.31931370.30041209ELECTRON CRYSTALLOGRAPHY80.89
2.77-2.910.39481400.29731272ELECTRON CRYSTALLOGRAPHY83.8
2.91-3.090.32431380.26341263ELECTRON CRYSTALLOGRAPHY82.7
3.09-3.330.26821410.25041255ELECTRON CRYSTALLOGRAPHY83.54
3.33-3.670.281430.20211283ELECTRON CRYSTALLOGRAPHY85.19
3.67-4.20.21931430.17711299ELECTRON CRYSTALLOGRAPHY86.09
4.2-5.280.19391390.15471289ELECTRON CRYSTALLOGRAPHY85.05
5.28-30.420.18261450.19461226ELECTRON CRYSTALLOGRAPHY81.41

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more