[English] 日本語
Yorodumi
- EMDB-8727: The cryo-ET structure of frozen-hydrated honey bee Z-disk -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-8727
TitleThe cryo-ET structure of frozen-hydrated honey bee Z-disk
Map datafrozen-hydrated honey bee Z-disk- filtered to 60 Angstrom
Sample
  • Organelle or cellular component: Z-disc
    • Protein or peptide: Actin
    • Protein or peptide: alpha-actinin
Biological speciesApis mellifera (honey bee)
Methodsubtomogram averaging / cryo EM / Resolution: 60.0 Å
AuthorsTaylor KA / Hu Z
Funding supportEuropean Union, United States, 3 items
OrganizationGrant numberCountry
European Communitys Seventh Framework Programme238423European Union
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM30598 United States
American Heart Association15PRE25090150 United States
CitationJournal: J Muscle Res Cell Motil / Year: 2017
Title: Structure of isolated Z-disks from honeybee flight muscle.
Authors: Mara Rusu / Zhongjun Hu / Kenneth A Taylor / John Trinick /
Abstract: The Z-disk is a complex structure comprising some 40 proteins that are involved in the transmission of force developed during muscle contraction and in important signalling pathways that govern ...The Z-disk is a complex structure comprising some 40 proteins that are involved in the transmission of force developed during muscle contraction and in important signalling pathways that govern muscle homeostasis. In the Z-disk the ends of antiparallel thin filaments from adjacent sarcomeres are crosslinked by α-actinin. The structure of the Z-disk lattice varies greatly throughout the animal kingdom. In vertebrates the thin filaments form a tetragonal lattice, whereas invertebrate flight muscle has a hexagonal lattice. The width of the Z-disk varies considerably and correlates with the number of α-actinin bridges. A detailed description at a high resolution of the Z-disk lattice is needed in order to better understand muscle function and disease. The molecular architecture of the Z-disk lattice in honeybee (Apis mellifera) is known from plastic embedded thin sections to a resolution of 7 nm, which is not sufficient to dock component protein crystal structures. It has been shown that sectioning is a damaging process that leads to the loss of finer details present in biological specimens. However, the Apis Z-disk is a thin structure (120 nm) suitable for cryo EM. We have isolated intact honeybee Z-disks from indirect flight muscle, thus obviating the need of plastic sectioning. We have employed cryo electron tomography and image processing to investigate the arrangement of proteins within the hexagonal lattice of the Apis Z-disk. The resolution obtained, ~6 nm, was probably limited by damage caused by the harshness of the conditions used to extract the myofibrils and isolate the Z-disks.
History
DepositionMay 7, 2017-
Header (metadata) releaseJun 14, 2017-
Map releaseAug 9, 2017-
UpdateAug 30, 2023-
Current statusAug 30, 2023Processing site: RCSB / Status: Released

-
Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0976
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.0976
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8727.png

Downloads & links

-
Map

FileDownload / File: emd_8727.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfrozen-hydrated honey bee Z-disk- filtered to 60 Angstrom
Voxel sizeX=Y=Z: 7.6 Å
Density
Contour LevelBy AUTHOR: 0.0876 / Movie #1: 0.0976
Minimum - Maximum-0.27998948 - 0.2915187
Average (Standard dev.)-0.000000000009657 (±0.072596684)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-96-96-96
Dimensions192192192
Spacing192192192
CellA=B=C: 1459.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z7.67.67.6
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z1459.2001459.2001459.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-210-210-210
NX/NY/NZ420420420
MAP C/R/S123
start NC/NR/NS-96-96-96
NC/NR/NS192192192
D min/max/mean-0.2800.292-0.000

-
Supplemental data

-
Additional map: frozen-hydrated honey bee Z-disk - raw tomogram. The...

Fileemd_8727_additional.map
Annotationfrozen-hydrated honey bee Z-disk - raw tomogram. The contour level here is non-sense. The map is raw tomogram, which is used to make the subvolume. Layer line data is not actually layer line, instead, it is a coordiates file for extracting subvolume
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Additional map: frozen-hydrated honey bee Z-disk - raw tomogram. The...

Fileemd_8727_additional_1.map
Annotationfrozen-hydrated honey bee Z-disk - raw tomogram. The contour level here is non-sense. The map is raw tomogram, which is used to make the subvolume. Layer line data is not actually layer line, instead, it is a coordiates file for extracting subvolume
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : Z-disc

EntireName: Z-disc
Components
  • Organelle or cellular component: Z-disc
    • Protein or peptide: Actin
    • Protein or peptide: alpha-actinin

-
Supramolecule #1: Z-disc

SupramoleculeName: Z-disc / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Z-disc directly isolated from Apis mellifera (honey bee) indirect flight muscle
Source (natural)Organism: Apis mellifera (honey bee) / Organ: flight muscle / Tissue: muscle / Organelle: Z-disc / Location in cell: sarcomere

-
Macromolecule #1: Actin

MacromoleculeName: Actin / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Apis mellifera (honey bee) / Organ: flight muscle / Tissue: muscle
SequenceString: MVGMGQKDSY VGDEAQSKRG ILTLKYPIEH GIITNWDDME KIWHHTFYNE LRVAPEEHPV LLTEAPLNPK ANREKMTQIM FETFNSPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVSHTV PIYEGYALPH AILRLDLAGR DLTDYLMKIL TERGYSFTTT AEREIVRDIK ...String:
MVGMGQKDSY VGDEAQSKRG ILTLKYPIEH GIITNWDDME KIWHHTFYNE LRVAPEEHPV LLTEAPLNPK ANREKMTQIM FETFNSPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVSHTV PIYEGYALPH AILRLDLAGR DLTDYLMKIL TERGYSFTTT AEREIVRDIK EKLCYVALDF EQEMATAAAS TSLEKSYELP DGQVITIGNE RFRCPEALFQ PSFLGMESCG IHETVYNSIM KCDVDIRKDL YANNVLSGGT TMYPGIADRM QKEITALAPS TIKIKIIAPP ERKYSVWIGG SILASLSTFQ QMWISKQEYD ESGPGIVHRK CF

-
Macromolecule #2: alpha-actinin

MacromoleculeName: alpha-actinin / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Apis mellifera (honey bee) / Organ: Flight muscle / Tissue: muscle
SequenceString: MEEYERLASD LLEWIRRTMP WLASRQTDNS LAGCQKKLEE YRTYRRKHKP PRVEQKAKLE TNFNTLQTKL RLSNRPAYMP TEGKMVSDIN KAWKGLELAE KSFEEWLLSE MMRLERLEHL AQKFKHKADA HEEWTAGKEE MLTSQHFRQC KLNELKALKK KHEAFESDLA ...String:
MEEYERLASD LLEWIRRTMP WLASRQTDNS LAGCQKKLEE YRTYRRKHKP PRVEQKAKLE TNFNTLQTKL RLSNRPAYMP TEGKMVSDIN KAWKGLELAE KSFEEWLLSE MMRLERLEHL AQKFKHKADA HEEWTAGKEE MLTSQHFRQC KLNELKALKK KHEAFESDLA AHQDRVEQIA AIAQELNTLE YHDSASVNAR CQRICDQWDR LGTLTQRRRQ ALDEAERILE KIDVLHLEFA KRAAPFNNWL DGTREDLVDM FIVHTMEEIQ GLMDAHAAFK ATLGEADKEY NAIVGLVREV ESIVKQFQIP GGLENPYTTL TALDLTKKWS DVRQLVPQRD GTLQAELRKQ QNNELLRRQF AEKANAVGPW IERQLDAVTA IGLGLQGTLE DQLHRLKEYE QAVYQYKVHL EELEKIHQAV QEGMIFENRY TQYTMETLRV GWEQLLTSIN RNINEVENQI LTRDSKGITQ EQLNEFRSSF NHFDKNRTGR LAPDEFKSCL VSLGYSIGKD RQGDIDFQRI LAIVDPNNSG YVHFDAFLDF MTRESTDTDT AEQVIDSFRI LAGDKPYILA DELRRELPPD QAEYCIQRMP PYKGPNAIPG ALDYRSFSTA LYGESDL

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statetissue

-
Sample preparation

BufferpH: 7.2
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
100.0 mMNaClSodium chlorideSodium Chloride
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: OTHER
Details: Carbon coated grids were made hydrophilic by glow discharging for 40 seconds at a high tension of 10 kV in a Cressington 208 Carbon Coater.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: After the Z-disk suspension was added to the grid it was left to settle for 10-15 seconds followed by washing with low salt buffer (25 mM HEPES, 100 mM NaCl). The washing step is crucial for ...Details: After the Z-disk suspension was added to the grid it was left to settle for 10-15 seconds followed by washing with low salt buffer (25 mM HEPES, 100 mM NaCl). The washing step is crucial for the removal of salts present in the extraction buffer, ensuring that plunge frozen grids were free of contamination. Plunge freezing was carried out using Quantifoil grids (Agar Scientific) in a Vitrobot Mark IV (FEI) at 4-5 degrees C, 90-100% humidity using a blotting force of 3-5 for 4-6 seconds..
DetailsIndirect flight muscle was harvested from the thorax of Apis mellifera obtained from a local bee keeper. Myofibrils in suspension were prepared according to previously published methods (Bullard et al., 1973; Saide and Ullrick, 1974) with the following modifications. The IFM were collected in ice cold sucrose containing buffer (0.3 M sucrose, 0.1 M KCl, 0.01 M potassium phosphate pH 7, 1 mM MgCl2, 2 mM EGTA, 0.02 M NaN3 and EDTA-free Protease Inhibitor Cocktail Tablets followed by homogenization using a Wheaton tissue grinder. Soluble proteins and sucrose were washed away by centrifugation in 0.1 M KCl, 0.01 M potassium phosphate pH 7 buffer. Myofibrils in suspension were stored in 75% glycerol at -80 degrees C. Intact Z-disks were isolated by incubating myofibrils on ice in a high ionic strength extraction solution containing 0.7 M KCl, 0.6 M KI, 0.08 M NaHCO3 pH 8 for 60 minutes. Following extraction Z-disks were either negatively stained or plunge frozen for further electron microscopic investigation.

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 22500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsTilt series were recorded using the Saxton acquisition scheme (Saxton et al., 1984). Tilt angles ranged from -69.87 to +67.66 degrees starting at 0 degrees, 96 total images, with an initial step of 2 degrees. The average electron dose/micrograph was ~0.7 electrons/Angstrom2. A total of 12 tilt series, designated tomo1 - tomo12, were collected but only one proved to be useful, tomo9. The others had poor resolution due to either low intrinsic order, excessive extraction of actin or other structural Z-disk proteins, or due to excessive radiation damage.
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 1.0 e/Å2 / Details: Electron dose may not be accurate.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

-
Image processing

Crystal parametersUnit cell - A: 520 Å / Unit cell - B: 520 Å / Unit cell - C: 425 Å / Unit cell - C sampling length: 950 Å / Unit cell - γ: 60 ° / Plane group: P 3 1 2
ExtractionNumber tomograms: 1 / Number images used: 399 / Reference model: cross correlation / Method: lattice fitting / Software - Name: I3 (ver. 0.9.4)
Details: Lattice positions were determined from a cross correlation map computed between the tomogram and a reference masked from within it. The Fourier transform was first filtered using a ...Details: Lattice positions were determined from a cross correlation map computed between the tomogram and a reference masked from within it. The Fourier transform was first filtered using a reciprocal lattice that contained 3 orders in a* and 5 orders in b*. Lattice positions were determined by peak fitting along a grid of predicted positions. Points on the predicted grid that fell outside of the actual Z-disk were removed as were any points that occurred too close to the edge of the tomogram. In total we extracted 399 subvolumes.
Final 3D classificationNumber classes: 1 / Avg.num./class: 399 / Details: Here only shows result of global average.
Final angle assignmentType: NOT APPLICABLE
Final reconstructionNumber classes used: 1 / Algorithm: EXACT BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 60.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: I3 (ver. 0.94)
Details: Base on structure feature, and the fitting model, the resolution was estimated manually. Also, the FSC yielded a value of 60 A.
Number subtomograms used: 399
FSC plot (resolution estimation)

-
Atomic model buiding 1

DetailsFor actin, filaments of 28 subunits having the helical symmetry of 28/13 were constructed and maps computed using the software pdb2mrc. These maps were then placed manually within the Z-disk thin filaments and fit quantitatively using chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT

+
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