[English] 日本語
Yorodumi
- PDB-8cpc: 3D electron diffraction structure of Hen Egg-White Lysozyme from ... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 8cpc
Title3D electron diffraction structure of Hen Egg-White Lysozyme from nano-crystals obtained by high pressure freezing and cryo-sectioning
ComponentsLysozyme C
KeywordsHYDROLASE / nano-crystals
Function / homology
Function and homology information


Lactose synthesis / Antimicrobial peptides / Neutrophil degranulation / beta-N-acetylglucosaminidase activity / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / defense response to Gram-negative bacterium / killing of cells of another organism / defense response to Gram-positive bacterium ...Lactose synthesis / Antimicrobial peptides / Neutrophil degranulation / beta-N-acetylglucosaminidase activity / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / defense response to Gram-negative bacterium / killing of cells of another organism / defense response to Gram-positive bacterium / defense response to bacterium / endoplasmic reticulum / extracellular space / identical protein binding / cytoplasm
Similarity search - Function
Glycoside hydrolase, family 22, lysozyme / Glycoside hydrolase family 22 domain / Glycosyl hydrolases family 22 (GH22) domain signature. / Glycoside hydrolase, family 22 / C-type lysozyme/alpha-lactalbumin family / Glycosyl hydrolases family 22 (GH22) domain profile. / Alpha-lactalbumin / lysozyme C / Lysozyme-like domain superfamily
Similarity search - Domain/homology
Biological speciesGallus gallus (chicken)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / negative staining / cryo EM / Resolution: 2.91 Å
AuthorsMoriscot, C. / Schoehn, G. / Housset, D.
Funding support France, 2items
OrganizationGrant numberCountry
Agence Nationale de la Recherche (ANR)ANR-10-INBS-0005-02 France
Agence Nationale de la Recherche (ANR)ANR-17-EURE-0003 France
CitationJournal: Ultramicroscopy / Year: 2023
Title: High pressure freezing and cryo-sectioning can be used for protein structure determination by electron diffraction.
Authors: Christine Moriscot / Guy Schoehn / Dominique Housset /
Abstract: Electron diffraction of three-dimensional nanometer sized crystals has emerged since 2013 as an efficient technique to solve the structure of both small organic molecules and model proteins. However, ...Electron diffraction of three-dimensional nanometer sized crystals has emerged since 2013 as an efficient technique to solve the structure of both small organic molecules and model proteins. However, the major bottleneck of the technique when applied to protein samples is to produce nano-crystals that do not exceed 200 to 300 nm in at least one dimension and to deposit them on a grid while keeping the minimum amount of solvent around them. Since the presence of amorphous solvent around the crystal, necessary to preserve its integrity, increases the amount of diffuse scattering, thus degrading the signal-to noise ratio of the diffraction signal, other sample preparation strategies have been developed. One of them is the milling of thin crystal lamella using focused ion beam (FIB), which was successfully applied to several protein crystals. Here, we present a new approach that uses cryo-sectioning after high pressure freezing of dextran embedded protein crystals. 150 to 200 nm thick cryo-sections of hen egg white lysozyme tetragonal crystals where used for electron diffraction experiments. Complete diffraction data up to 2.9 Å resolution have been collected and the lysozyme structure has been solved by molecular replacement and refined against these data. Our data demonstrate that cryo-sectioning can preserve protein structure at high resolution and can be used as a new sample preparation technique for 3D electron diffraction experiments of protein crystals. The different orientations found in the crystal chips and their large number resulting from the cryo-sectioning make the latter an attractive approach as it combines advantages from both blotting approaches (number of crystals) and FIB-milling (controlled thickness and absence of solvent around the crystal).
History
DepositionMar 2, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 20, 2023Provider: repository / Type: Initial release

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Lysozyme C


Theoretical massNumber of molelcules
Total (without water)14,3311
Polymers14,3311
Non-polymers00
Water1086
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area6640 Å2
MethodPISA
Unit cell
Length a, b, c (Å)76.887, 76.887, 37.761
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Components on special symmetry positions
IDModelComponents
11A-204-

HOH

-
Components

#1: Protein Lysozyme C / 1 / 4-beta-N-acetylmuramidase C / Allergen Gal d IV


Mass: 14331.160 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / References: UniProt: P00698, lysozyme
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: Monomer of Hen Egg-White Lysozyme / Type: COMPLEX / Details: Purchased / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
EM crystal formationDetails: Lysozyme (50 mg/ml) was mixed 1 to 1 to precipitant solution (100mM Na citrate pH 5.5 and 0.8 to 1.6 M NaCl). Crystals were grown by the hanging drop method at room temperature.
Buffer solutionpH: 5.5 / Details: 100 mM sodium citrate pH 5.5, 0.8-1.6 M NaCl
Buffer component
IDConc.NameBuffer-ID
10.1 Msodium citrate1
21.2 Msodium chloride1
SpecimenConc.: 50 mg/ml / Embedding applied: YES / Shadowing applied: NO / Staining applied: YES / Vitrification applied: YES
EM stainingType: NONE
Details: The blue dye (Brillant blue) is used to see the crystals during cryo-sectioning
Material: Brilliant blue
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil
EM embeddingDetails: 40% Dextran in phosphate buffer / Material: dextran
VitrificationCryogen name: NITROGEN
Details: Freezing carried out at high pressure (210 MPa) and -196 C, using a Leica EM HPM100

-
Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Details: Camera length used 730 mm and 1000 mm (corresponding to 1285 and 1825 mm calibrated values)
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Temperature (max): 106 K / Temperature (min): 101 K
Image recordingAverage exposure time: 0.25 sec. / Electron dose: 0.004 e/Å2 / Film or detector model: OTHER / Num. of diffraction images: 1408 / Num. of grids imaged: 1
Details: ASI Medipix3RX hybrid pixel detector used. Images were collected at 4 frames per second, in continuous rotation mode and 0.367 degrees per frames.
Image scansSampling size: 55 µm / Width: 512 / Height: 512
EM diffraction shell
Resolution (Å)IDEM diffraction stats-IDFourier space coverage (%)MultiplicityNum. of structure factorsPhase residual (°)
2.91-54.371196.533.126400.001
2.91-2.992179.611.81560.001
EM diffraction statsDetails: Phases obtained by Molecular Replacement, using PHASER and the 193L pdb entry as model.
Fourier space coverage: 96.5 % / High resolution: 2.91 Å / Num. of intensities measured: 87489 / Num. of structure factors: 2640 / Phase error rejection criteria: no phase error rejection / Rmerge: 61.1

-
Processing

SoftwareName: REFMAC / Version: 5.8.0267 / Classification: refinement / Contact author: Garib N. Murshudov / Contact author email: garib[at]mrc-lmb.cam.ac.uk / Date: 2020-24-08
Description: (un)restrained refinement or idealisation of macromolecular structures
EM softwareVersion: 2.8.3 / Category: molecular replacement
Image processingDetails: A flat-field correction was applied to all diffraction images with ImageJ before data processing with XDS.
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 76.89 Å / B: 76.89 Å / C: 37.76 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionMethod: CRYSTALLOGRAPHY / Resolution: 2.91 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Details: Diffraction data were processed with XDS and merged with XSCALE. The structure was solved by molecular replacement with PHASER 2.8.3 and refined with Refmac 5.8.0267, using electron atomic ...Details: Diffraction data were processed with XDS and merged with XSCALE. The structure was solved by molecular replacement with PHASER 2.8.3 and refined with Refmac 5.8.0267, using electron atomic scattering factors (source electron used in Refmac).
Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER
RefinementResolution: 2.91→54.367 Å / Cor.coef. Fo:Fc: 0.928 / Cor.coef. Fo:Fc free: 0.839 / SU B: 25.859 / SU ML: 0.451 / Cross valid method: FREE R-VALUE / ESU R Free: 0.591
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2949 276 10.462 %
Rwork0.2048 2362 -
all0.214 --
obs-2638 96.418 %
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 31.961 Å2
Baniso -1Baniso -2Baniso -3
1--0.063 Å20 Å20 Å2
2---0.063 Å20 Å2
3---0.125 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0050.0131034
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0010.014950
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.4891.6361402
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.2051.5952163
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg8.3685130
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg32.42820.31763
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg21.07715169
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg17.7841512
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0590.2131
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0050.021222
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0010.02286
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2250.2239
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_other0.1880.2947
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.1690.2489
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbtor_other0.0780.2497
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.1580.222
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.1170.27
ELECTRON CRYSTALLOGRAPHYr_nbd_other0.160.234
ELECTRON CRYSTALLOGRAPHYr_mcbond_it2.1653.341517
ELECTRON CRYSTALLOGRAPHYr_mcbond_other2.1023.324516
ELECTRON CRYSTALLOGRAPHYr_mcangle_it3.7974.996645
ELECTRON CRYSTALLOGRAPHYr_mcangle_other3.8195.012646
ELECTRON CRYSTALLOGRAPHYr_scbond_it1.8973.486517
ELECTRON CRYSTALLOGRAPHYr_scbond_other1.8753.483517
ELECTRON CRYSTALLOGRAPHYr_scangle_it3.415.177756
ELECTRON CRYSTALLOGRAPHYr_scangle_other3.4095.174756
ELECTRON CRYSTALLOGRAPHYr_lrange_it6.52437.4691198
ELECTRON CRYSTALLOGRAPHYr_lrange_other6.52137.5511199
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
2.91-2.9850.329180.3511360.3481950.6010.64878.97440.34
2.985-3.0660.388230.3451480.3511920.6760.72789.06250.319
3.066-3.1550.523180.2881570.3111870.5780.80393.58290.265
3.155-3.2520.241200.2831380.2771750.8320.84190.28570.242
3.252-3.3580.347170.2461570.2571740.8680.8751000.209
3.358-3.4750.353130.2151580.2231710.8320.9111000.177
3.475-3.6060.411170.2051420.231600.8780.90699.3750.182
3.606-3.7530.294210.2191440.2281650.8980.9211000.196
3.753-3.9190.146140.1941350.191500.950.93699.33330.169
3.919-4.1090.338140.1821320.1961460.9130.9431000.149
4.109-4.330.33480.1591310.1711400.9230.95899.28570.136
4.33-4.590.14140.1661200.1641340.9510.9621000.144
4.59-4.9050.228130.1311140.1421280.9530.96799.21880.111
4.905-5.2940.265150.1471040.1611190.940.9641000.11
5.294-5.7940.349100.171990.1841090.8840.9571000.135
5.794-6.4690.295100.164950.1741050.9220.9661000.131
6.469-7.4520.176110.193790.19900.950.9481000.158
7.452-9.0840.58940.193780.203820.7590.9621000.15
9.084-12.6720.293120.229570.242690.9460.961000.209
12.672-54.3670.11640.193380.188450.9560.97393.33330.156

+
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://pdbjlvh1.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