[English] 日本語
Yorodumi
- PDB-6n4u: MicroED structure of Proteinase K at 2.75A resolution from a sing... -

+
Open data


ID or keywords:

Loading...

no data

-
Basic information

Entry
Database: PDB / ID: 6n4u
TitleMicroED structure of Proteinase K at 2.75A resolution from a single milled crystal.
ComponentsProteinase K
KeywordsHYDROLASE / broad-spectrum serum proteinase
Function / homologyProteinase K-like catalytic domain / Serine proteases, subtilase family, aspartic acid active site. / Serine proteases, subtilase family, serine active site. / 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 / Serine proteases, subtilase family, histidine active site. ...Proteinase K-like catalytic domain / Serine proteases, subtilase family, aspartic acid active site. / Serine proteases, subtilase family, serine active site. / 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 / Serine proteases, subtilase family, histidine active site. / Peptidase S8/S53 domain superfamily / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Subtilase family / Peptidase inhibitor I9 / peptidase K / serine-type endopeptidase activity / metal ion binding / Proteinase K
Function and homology information
Specimen sourceEngyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / 2.75 Å resolution
AuthorsMartynowycz, M.W. / Zhao, W. / Hattne, J. / Jensen, G.J. / Gonen, T.
CitationJournal: Structure / Year: 2018
Title: Collection of Continuous Rotation MicroED Data from Ion Beam-Milled Crystals of Any Size.
Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen
Abstract: Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation ...Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoelectron microscopy as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to desired dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 20, 2018 / Release: Feb 6, 2019

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-0343
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,1354
Polyers28,9591
Non-polymers1763
Water41423
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)260
ΔGint (kcal/M)-21
Surface area (Å2)10120
Unit cell
γ
α
β
Length a, b, c (Å)67.460, 67.460, 106.670
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP 43 21 2

-
Components

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


Mass: 28958.791 Da / Num. of mol.: 1 / Source: (gene. exp.) Engyodontium album (fungus) / Gene: PROK / Production host: Engyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Formula: Ca / Calcium
#3: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Formula: SO4 / Sulfate
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 23 / Formula: H2O / Water

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: Proteinase K / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: 1 / Source: NATURAL
Molecular weightValue: 0.02893 MDa / Experimental value: NO
Source (natural)Organism: Engyodontium album (fungus)
Buffer solutionpH: 7.5
SpecimenConc.: 20 mg/ml / Details: Proteinase K purchased from Sigma. / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 273 kelvins

-
Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / C2 aperture diameter: 100 microns / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 kelvins / Temperature (min): 77 kelvins
Image recordingAverage exposure time: 2 sec. / Electron dose: 0.04 e/Å2
Details: Continuous rotation from -30 to +30 with a rotation rate of 0.2 degrees per second and a readout every 3 seconds.
Film or detector model: FEI CETA (4k x 4k) / Number of diffraction images: 100 / Number of grids imaged: 1
Image scansSampling size: 28 microns / Width: 4096 / Height: 4096
EM diffractionCamera length: 1700 mm
EM diffraction shellFourier space coverage: 88.23 / High resolution: 2.75 Å / Low resolution: 3.1483 Å / Multiplicity: 2.9 / Number of structure factors: 1831 / Phase residual: 28.11 deg.
EM diffraction statsFourier space coverage: 88.4 / High resolution: 2.75 Å / Number of intensities measured: 31013 / Number of structure factors: 10807 / Overall phase error: 22.41 deg. / Overall phase residual: 22.41 deg. / Phase error rejection criteria: 0 / R merge: 0.47 / R sym: 0.39

-
Processing

SoftwareName: PHENIX / Version: (1.14_3260: ???) / Classification: refinement
EM software
IDNameVersionCategory
6PHENIX1.14model fitting
8PHENIX2.8.2molecular replacement
13PHENIX1.14model refinement
Image processingDetails: This was the new CetaD.
EM 3D crystal entityAngle alpha: 90 deg. / Angle beta: 90 deg. / Angle gamma: 90 deg. / Length a: 67.46 Å / Length b: 67.46 Å / Length c: 106.67 Å / Space group name: P43 21 2 / Space group num: 96
CTF correctionDetails: NONE / Type: NONE
3D reconstructionResolution: 2.75 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingOverall b value: 21.1 / Ref protocol: RIGID BODY FIT / Ref space: RECIPROCAL / Target criteria: R
Atomic model buildingPDB-ID: 5I9S
RefineMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6CL7
Overall SU ML: 0.36 / R Free selection details: From 5I9S / Cross valid method: FREE R-VALUE / Sigma F: 1.34 / Overall phase error: 24.25
Solvent computationSolvent shrinkage radii: 0.9 Å / Solvent vdw probe radii: 1.11 Å
Least-squares processR factor R free: 0.2626 / R factor R work: 0.2377 / R factor obs: 0.2393 / Highest resolution: 2.75 Å / Lowest resolution: 43.546 Å / Number reflection R free: 383 / Number reflection obs: 6008 / Percent reflection R free: 6.37 / Percent reflection obs: 87.76
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0052072
ELECTRON CRYSTALLOGRAPHYf_angle_d0.6972818
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d14.908720
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.044312
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.004370
Refine LS shell

Refine ID: ELECTRON CRYSTALLOGRAPHY

Highest resolutionR factor R freeR factor R workLowest resolutionNumber reflection R freeNumber reflection R workPercent reflection obs
2.75040.33620.30323.1483124183189.00
3.14830.25600.24083.9661126187088.00
3.96610.23310.204743.5511133192487.00

+
About Yorodumi

-
News

-
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. (see PDBe EMDB page)
  • 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 at EBI / 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. See below links for details.
  • 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

+
Jun 16, 2017. Omokage search with filter

Omokage search with filter

  • Result of Omokage search can be filtered by keywords and the database types

Related info.: Omokage search

+
Sep 15, 2016. EM Navigator & Yorodumi renewed

EM Navigator & Yorodumi renewed

  • New versions of EM Navigator and Yorodumi started

Related info.: Changes in new EM Navigator and Yorodumi

+
Aug 31, 2016. New EM Navigator & Yorodumi

New EM Navigator & Yorodumi

  • In 15th Sep 2016, the development versions of EM Navigator and Yorodumi will replace the official versions.
  • Current version will continue as 'legacy version' for some time.

Related info.: Changes in new EM Navigator and Yorodumi / EM Navigator / Yorodumi

Read more

-
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.

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