[English] 日本語
Yorodumi
- EMDB-20366: MicroED structure of proteinase K from merging low-dose, platinum... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-20366
TitleMicroED structure of proteinase K from merging low-dose, platinum pre-coated lamellae at 1.85A resolution (LDPT)
Map dataMicroED 2Fo-Fc map from low-dose merged, platinum-coated, lamellae at 1.85A resolution (LDPT)
Sample
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: water
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. ...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 / Subtilase family / Peptidase S8/S53 domain
Similarity search - Domain/homology
Biological speciesParengyodontium album (fungus)
Methodelectron crystallography / cryo EM / Resolution: 1.85 Å
AuthorsMartynowycz MW / Zhao W
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R35 GM122588 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)2P50GM082545 United States
CitationJournal: Structure / Year: 2019
Title: Qualitative Analyses of Polishing and Precoating FIB Milled Crystals for MicroED.
Authors: Michael W Martynowycz / Wei Zhao / Johan Hattne / Grant J Jensen / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction ...Microcrystal electron diffraction (MicroED) leverages the strong interaction between matter and electrons to determine protein structures from vanishingly small crystals. This strong interaction limits the thickness of crystals that can be investigated by MicroED, mainly due to absorption. Recent studies have demonstrated that focused ion-beam (FIB) milling can thin crystals into ideal-sized lamellae; however, it is not clear how to best apply FIB milling for MicroED. Here, the effects of polishing the lamellae, whereby the last few nanometers are milled away using a low-current gallium beam, are explored in both the platinum-precoated and uncoated samples. Our results suggest that precoating samples with a thin layer of platinum followed by polishing the crystal surfaces prior to data collection consistently led to superior results as indicated by higher signal-to-noise ratio, higher resolution, and better refinement statistics. This study lays the foundation for routine and reproducible methodology for sample preparation in MicroED.
History
DepositionJun 29, 2019-
Header (metadata) releaseJul 24, 2019-
Map releaseSep 4, 2019-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.482265
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.482265
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6pkt
  • Surface level: 0.482265
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20366.png

Downloads & links

-
Map

FileDownload / File: emd_20366.map.gz / Format: CCP4 / Size: 32.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMicroED 2Fo-Fc map from low-dose merged, platinum-coated, lamellae at 1.85A resolution (LDPT)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.33 Å/pix.
x 211 pix.
= 70.421 Å		0.35 Å/pix.
x 201 pix.
= 70.434 Å		0.35 Å/pix.
x 201 pix.
= 70.434 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX: 0.35042 Å / Y: 0.35042 Å / Z: 0.33375 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.482265 / Movie #1: 0.482265
Minimum - Maximum-0.7197126 - 1.96934
Average (Standard dev.)-0.000011349287 (±0.32151476)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-39-149-104
Dimensions201201211
Spacing201201211
CellA: 70.43375 Å / B: 70.43375 Å / C: 70.42125 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.350417910447760.350417910447760.33374881516588
M x/y/z201201211
origin x/y/z0.0000.0000.000
length x/y/z70.43470.43470.421
α/β/γ90.00090.00090.000
start NX/NY/NZ-39-149-104
NX/NY/NZ201201211
MAP C/R/S213
start NC/NR/NS-149-39-104
NC/NR/NS201201211
D min/max/mean-0.7201.969-0.000

-
Supplemental data

-
Sample components

-
Entire : Proteinase K

EntireName: Proteinase K
Components
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: water

-
Supramolecule #1: Proteinase K

SupramoleculeName: Proteinase K / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Parengyodontium album (fungus)
Molecular weightTheoretical: 28.93 KDa

-
Macromolecule #1: Proteinase K

MacromoleculeName: Proteinase K / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: peptidase K
Source (natural)Organism: Parengyodontium album (fungus)
Molecular weightTheoretical: 28.930783 KDa
SequenceString: AAQTNAPWGL ARISSTSPGT STYYYDESAG QGSCVYVIDT GIEASHPEFE GRAQMVKTYY YSSRDGNGHG THCAGTVGSR TYGVAKKTQ LFGVKVLDDN GSGQYSTIIA GMDFVASDKN NRNCPKGVVA SLSLGGGYSS SVNSAAARLQ SSGVMVAVAA G NNNADARN ...String:
AAQTNAPWGL ARISSTSPGT STYYYDESAG QGSCVYVIDT GIEASHPEFE GRAQMVKTYY YSSRDGNGHG THCAGTVGSR TYGVAKKTQ LFGVKVLDDN GSGQYSTIIA GMDFVASDKN NRNCPKGVVA SLSLGGGYSS SVNSAAARLQ SSGVMVAVAA G NNNADARN YSPASEPSVC TVGASDRYDR RSSFSNYGSV LDIFGPGTSI LSTWIGGSTR SISGTSMATP HVAGLAAYLM TL GKTTAAS ACRYIADTAN KGDLSNIPFG TVNLLAYNNY QA

UniProtKB: Proteinase K

-
Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 150 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

-
Experimental details

-
Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state3D array

-
Sample preparation

Concentration20 mg/mL
BufferpH: 7.5
GridSupport film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Details: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 273.0 K / Instrument: FEI VITROBOT MARK IV
DetailsProteinase K purchased from Sigma.

-
Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 77.0 K / Max: 90.0 K
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 100 / Number diffraction images: 100 / Average exposure time: 3.0 sec. / Average electron dose: 0.03 e/Å2
Details: Continuous rotation with a rotation rate of 0.2 degrees per second and a readout every 3 seconds
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 2055.0 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

-
Image processing

DetailsThis was the new CetaD.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 1.85 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Software - Name: PHENIX (ver. 1.15.2)
Molecular replacementSoftware - Name: PHENIX (ver. 2.8.2)
Crystallography statisticsNumber intensities measured: 150310 / Number structure factors: 19710 / Fourier space coverage: 90.75 / R sym: 0.26 / R merge: 0.24 / Overall phase error: 22.57 / Overall phase residual: 22.57 / Phase error rejection criteria: 0 / High resolution: 1.85 Å / Shell - Shell ID: 1 / Shell - High resolution: 1.85 Å / Shell - Low resolution: 32.08 Å / Shell - Number structure factors: 19710 / Shell - Phase residual: 22.57 / Shell - Fourier space coverage: 90.75 / Shell - Multiplicity: 7.6

-
Atomic model buiding 1

Initial modelPDB ID:

6cl7


Chain - Chain ID: A / Chain - Residue range: 106-384 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 24
Output model

PDB-6pkt:
MicroED structure of proteinase K from merging low-dose, platinum pre-coated lamellae at 1.85A resolution (LDPT)

+
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