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

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Basic information

Entry
Database: PDB / ID: 6iug
TitleCryo-EM structure of the plant actin filaments from Zea mays pollen
Componentspollen F-actin
KeywordsPROTEIN FIBRIL / Microfilament / Helix / Actin
Function / homology
Function and homology information


cytoskeleton / ATP binding / cytoplasm
Similarity search - Function
ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / Actins signature 1. / Actin, conserved site / Actins signature 2. / Actin/actin-like conserved site / Actins and actin-related proteins signature. / Actin / Actin family ...ATPase, substrate binding domain, subdomain 4 / Actin; Chain A, domain 4 / ATPase, nucleotide binding domain / 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 / Nucleotidyltransferase; domain 5 / Alpha-Beta Complex / 2-Layer Sandwich / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / Actin-1
Similarity search - Component
Biological speciesZea mays (maize)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsRen, Z.H. / Zhang, Y. / Zhang, Y. / He, Y.Q. / Du, P.Z. / Wang, Z.X. / Sun, F. / Ren, H.Y.
Funding support China, 5items
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, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 25, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title
Revision 1.2Mar 27, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

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  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: pollen F-actin
B: pollen F-actin
C: pollen F-actin
D: pollen F-actin
E: pollen F-actin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,31315
Polymers206,0565
Non-polymers2,25810
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15130 Å2
ΔGint-133 kcal/mol
Surface area74470 Å2

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Components

#1: Protein
pollen F-actin


Mass: 41211.121 Da / Num. of mol.: 5 / Source method: isolated from a natural source / Source: (natural) Zea mays (maize) / Organ: pollen grains
Plasmid details: The cultivar of maize (Zea mays) plants is longping No.206.
References: UniProt: B6TQ08
#2: Chemical
ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE / Adenosine diphosphate


Mass: 427.201 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: The actin filament from Zea mays pollen / Type: COMPLEX
Details: Plant actin was purified from the pollen which was collected from maize (Zea mays) plants and polymerized at room temperature for 4 hours.
Entity ID: #1 / Source: NATURAL
Molecular weightValue: 15 kDa/nm
Source (natural)Organism: Zea mays (maize) / Organ: Pollen grains
Buffer solutionpH: 7
Buffer component
IDConc.FormulaBuffer-ID
15 mMTris1
20.01 %NaN31
30.5 mMDTT1
40.2 mMATPAdenosine triphosphate1
550 mMKCl1
61 mMMgCl21
70.8 mMEGTA1
810 mMimidazole1
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Plant 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.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K
Details: Blotting time of 5.5 s and blotting force of level 2

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 X / Calibrated magnification: 22013 X / Calibrated defocus min: 1200 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5.44 sec. / Electron dose: 39 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3605
Details: A total of 3,605 micrographs were recorded at a calibrated pixel size of 1.063 angstrom.
Image scansWidth: 7676 / Height: 7420 / Movie frames/image: 32 / Used frames/image: 2-27

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SPIDER24.08particle selectionSpider script was used to generate actin segments
3SerialEM3.5image acquisition
5Gctf1.18CTF correction
8UCSF Chimera1.10.1model fitting
9Coot7model fitting
11PHENIX1.11.1model refinement
12SPIDER24.08initial Euler assignment
13SPIDER24.08final Euler assignment
14SPIDER24.08classification
15SPIDER24.083D reconstruction
Image processingDetails: The 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.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -166.77 ° / Axial rise/subunit: 27.5 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 40943
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.
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 35684 / Algorithm: BACK PROJECTION / Num. of class averages: 5100 / Symmetry type: HELICAL
Atomic model buildingB value: 70 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Correlation coefficient
Details: Chain 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 ...Details: Chain 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.
Atomic model buildingPDB-ID: 5OOC

5ooc


Pdb chain-ID: C / Accession code: 5OOC / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00514910
ELECTRON MICROSCOPYf_angle_d0.89220235
ELECTRON MICROSCOPYf_dihedral_angle_d9.0818970
ELECTRON MICROSCOPYf_chiral_restr0.0552250
ELECTRON MICROSCOPYf_plane_restr0.0072580

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