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- EMDB-9734: Cryo-EM structure of the plant actin filaments from Zea mays pollen -

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Basic information

Entry
Database: EMDB / ID: EMD-9734
TitleCryo-EM structure of the plant actin filaments from Zea mays pollen
Map dataNone
Sample
  • Complex: The actin filament from Zea mays pollen
    • Protein or peptide: pollen F-actin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsMicrofilament / Helix / Actin / PROTEIN FIBRIL
Function / homology
Function and homology information


cytoskeleton / ATP binding
Similarity search - Function
Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family / Actin / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Biological speciesZea mays (maize)
Methodhelical reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsRen ZH / Zhang Y
Funding support China, 5 items
OrganizationGrant numberCountry
National Natural Science Foundation of China91854206 China
National Natural Science Foundation of China31770206 China
National Natural Science Foundation of China31770794 China
Ministry of Science and Technology (China)2013CB126902 China
Ministry of Science and Technology (China)2017YFA0504700 China
CitationJournal: Plant Cell / Year: 2019
Title: Cryo-EM Structure of Actin Filaments from Pollen.
Authors: Zhanhong Ren / Yan Zhang / Yi Zhang / Yunqiu He / Pingzhou Du / Zhanxin Wang / Fei Sun / Haiyun Ren /
Abstract: Actins are among the most abundant and conserved proteins in eukaryotic cells, where they form filamentous structures that perform vital roles in key cellular processes. Although large amounts of ...Actins are among the most abundant and conserved proteins in eukaryotic cells, where they form filamentous structures that perform vital roles in key cellular processes. Although large amounts of data on the biochemical activities, dynamic behaviors, and important cellular functions of plant actin filaments have accumulated, their structural basis remains elusive. Here, we report a 3.9 Å structure of the plant actin filament from pollen (ZMPA) using cryo-electron microscopy. The structure shows a right-handed, double-stranded (two parallel strands) and staggered architecture that is stabilized by intra- and interstrand interactions. While the overall structure resembles that of other actin filaments, its DNase I binding loop bends farther outward, adopting an open conformation similar to that of the jasplakinolide- or beryllium fluoride (BeF)-stabilized rabbit skeletal muscle actin (RSMA) filament. Single-molecule magnetic tweezers analysis revealed that the ZMPA filament can resist a greater stretching force than the RSMA filament. Overall, these data provide evidence that plant actin filaments have greater stability than animal actin filaments, which might be important to their role as tracks for long-distance vesicle and organelle transportation.plantcell;31/12/2855/FX1F1fx1.
History
DepositionNov 28, 2018-
Header (metadata) releaseNov 6, 2019-
Map releaseNov 6, 2019-
UpdateMar 27, 2024-
Current statusMar 27, 2024Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.000291
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.000291
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6iug
  • Surface level: 0.000291
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6iug
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9734.png

Downloads & links

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Map

FileDownload / File: emd_9734.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 200 pix.
= 212.6 Å		1.06 Å/pix.
x 200 pix.
= 212.6 Å		1.06 Å/pix.
x 200 pix.
= 212.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.063 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.000291 / Movie #1: 0.000291
Minimum - Maximum-0.00038637855 - 0.0008742896
Average (Standard dev.)-0.000039204464 (±0.00007096263)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-100-100-100
Dimensions200200200
Spacing200200200
CellA=B=C: 212.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0631.0631.063
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.600212.600212.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-100-100-100
NC/NR/NS200200200
D min/max/mean-0.0000.001-0.000

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Supplemental data

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Sample components

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Entire : The actin filament from Zea mays pollen

EntireName: The actin filament from Zea mays pollen
Components
  • Complex: The actin filament from Zea mays pollen
    • Protein or peptide: pollen F-actin
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: The actin filament from Zea mays pollen

SupramoleculeName: The actin filament from Zea mays pollen / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Plant actin was purified from the pollen which was collected from maize (Zea mays) plants and polymerized at room temperature for 4 hours.
Source (natural)Organism: Zea mays (maize) / Organ: Pollen grains
Molecular weightTheoretical: 15 kDa/nm

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Macromolecule #1: pollen F-actin

MacromoleculeName: pollen F-actin / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Zea mays (maize) / Organ: pollen grains
Molecular weightTheoretical: 41.211121 KDa
SequenceString: IQPLVCDNGT GMVKAGFAGD DAPRAVFPSI VGRPRHTGVM VGMGQKDAYV GDEAQSKRGI LTLKYPIEHG IVSNWDDMEK IWHHTFYNE LRVAPEEHPV LLTEAPLNPK ANREKMTQIM FETFNTPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVSHTV P IYEGYALP ...String:
IQPLVCDNGT GMVKAGFAGD DAPRAVFPSI VGRPRHTGVM VGMGQKDAYV GDEAQSKRGI LTLKYPIEHG IVSNWDDMEK IWHHTFYNE LRVAPEEHPV LLTEAPLNPK ANREKMTQIM FETFNTPAMY VAIQAVLSLY ASGRTTGIVL DSGDGVSHTV P IYEGYALP HAILRLDLAG RDLTDYLMKI LTERGYSFTT TAEREIVRDM KEKLAYIALD YDQEMETAKT SSSVEKSYEL PD GQVITIG AERFRCPEVL FQPSFIGMEA AGIHETTYNS IMKCDVDIRK DLYGNIVLSG GTTMFPGIAD RMSKEITALA PSS MKIKVV APPERKYSVW IGGSILASLS TFQQMWIAKA EYDESGPSIV HRKCF

UniProtKB: Actin-1

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Macromolecule #2: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.4 mg/mL
BufferpH: 7
Component:
ConcentrationFormula
5.0 mMTris
0.01 %NaN3
0.5 mMDTT
0.2 mMATP
50.0 mMKCl
1.0 mMMgCl2
0.8 mMEGTA
10.0 mMimidazole
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100.0 % / Chamber temperature: 298.0 K / Instrument: FEI VITROBOT MARK IV
Details: Blotting time of 5.5 s and blotting force of level 2.
DetailsPlant actin was dialyzed against buffer solution with pH 7.0 overnight in order to change the alkaline pH to the neutral pH and then polymerized at the final concentration of 0.4 mg/mL.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 2-27 / Number real images: 3605 / Average exposure time: 5.44 sec. / Average electron dose: 39.0 e/Å2
Details: A total of 3,605 micrographs were recorded at a calibrated pixel size of 1.063 angstrom.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 1.2 µm / Calibrated magnification: 22013 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe micrographs such as those with pollutions, bad Thon rings, large defocus values and others, were excluded before filament boxing. 1,540 micrographs were finally sorted out as "good" ones.
Final reconstructionNumber classes used: 5100
Applied symmetry - Helical parameters - Δz: 27.5 Å
Applied symmetry - Helical parameters - Δ&Phi: -166.77 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: SPIDER (ver. 24.08) / Number images used: 35684
Segment selectionNumber selected: 40943 / Software - Name: SPIDER (ver. 24.08)
Software - details: Spider script was used to generate actin segments
Details: A total of 8,609 ZMPA filaments were boxed using e2helixboxer.py in the package of EMAN2 with box width 168 and 77% box-overlap. A total of 40,943 segments were generated with box-size of 384.
Startup modelType of model: OTHER
Details: A cylinder with diamter the same as plant actin was generated by Spider and used as starting model
Final angle assignmentType: NOT APPLICABLE / Software - Name: SPIDER (ver. 24.08)
Details: Euler angles were assigned by cross correlation calcualtion of projection matching between projections of the model generated from last iteration and the images.

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Atomic model buiding 1

Initial modelPDB ID:

5ooc


Chain - Chain ID: C / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsChain C from pdb 5OOC was rigid body fitted to plant actin map using Chimera; Then Phenix was used for real space refinement. Finally, filament with 5 real space refinement units were generated using helical parameter, and this filament with 5 units was real space refined using Phenix again.
RefinementSpace: RECIPROCAL / Protocol: RIGID BODY FIT / Overall B value: 70 / Target criteria: Correlation coefficient
Output model

PDB-6iug:
Cryo-EM structure of the plant actin filaments from Zea mays pollen

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