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- PDB-9ikm: Cryo-EM structure of TLP-4 -

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Basic information

Entry
Database: PDB / ID: 9ikm
TitleCryo-EM structure of TLP-4
ComponentsTLP-4
KeywordsPROTEIN FIBRIL / Fibril
Biological speciesalgae metagenome (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsYan, N. / Yan, C. / Li, Z. / Wang, T.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32330052 China
National Natural Science Foundation of China (NSFC)32341016 China
National Natural Science Foundation of China (NSFC)32171204 China
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: CryoSeek II: Cryo-EM analysis of glycofibrils from freshwater reveals well-structured glycans coating linear tetrapeptide repeats.
Authors: Tongtong Wang / Wenze Huang / Kui Xu / Yitong Sun / Qiangfeng Cliff Zhang / Chuangye Yan / Zhangqiang Li / Nieng Yan /
Abstract: Despite the recent breakthrough in structure determination and prediction of proteins, the structural investigation of carbohydrates remains a challenge. Here, we report the cryo-EM analysis of a ...Despite the recent breakthrough in structure determination and prediction of proteins, the structural investigation of carbohydrates remains a challenge. Here, we report the cryo-EM analysis of a glycofibril found in the freshwater in the Tsinghua Lotus Pond. The fibril, which we name TLP-4, is made of a linear chain of tetrapeptide repeats coated with >4 nm thick glycans. In each repeat, two glycans are O-linked to a 3,4-dihydroxyproline and another glycan attaches to the adjacent Ser or Thr. The fibril structure is entirely maintained through glycan packing. Bioinformatic analysis confirms the conservation of the TLP-4 repeats across species, suggesting the existence of a large number of glycofibrils to be discovered. Our findings not only provide valuable insights into the structural roles of glycans in bio-assemblies but also demonstrate the potential of our recently formulated research strategy of CryoSeek to find bioentities and establish prototypes for structural studies of carbohydrates.
History
DepositionJun 27, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jan 8, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: TLP-4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,73055
Polymers7,0091
Non-polymers136,72154
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein TLP-4


Mass: 7008.729 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) algae metagenome (others)
#2: Polysaccharide
alpha-L-arabinofuranose-(1-2)-beta-D-galactopyranose-(1-2)-beta-L-arabinofuranose-(1-2)-[alpha-L- ...alpha-L-arabinofuranose-(1-2)-beta-D-galactopyranose-(1-2)-beta-L-arabinofuranose-(1-2)-[alpha-L-arabinofuranose-(1-2)-beta-L-arabinofuranose-(1-3)]beta-L-arabinofuranose-(1-3)-alpha-L-arabinofuranose


Type: oligosaccharide / Mass: 972.845 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LArafa1-2DGalpb1-2LArafb1-2[LArafa1-2LArafb1-3]LArafb1-3LArafa1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,7,6/[a211h-1a_1-4][a211h-1b_1-4][a2112h-1b_1-5]/1-2-2-3-1-2-1/a3-b1_b2-c1_b3-f1_c2-d1_d2-e1_f2-g1WURCSPDB2Glycan 1.1.0
[][L-1-deoxy-Araf]{[(3+1)][b-L-Araf]{[(2+1)][b-L-Araf]{[(2+1)][b-D-Galp]{[(2+1)][a-L-Araf]{}}}[(3+1)][b-L-Araf]{[(2+1)][a-L-Araf]{}}}}LINUCSPDB-CARE
#3: Polysaccharide
alpha-L-arabinofuranose-(1-3)-alpha-L-arabinofuranose-(1-3)-alpha-D-mannopyranose-(1-2)-[alpha-L- ...alpha-L-arabinofuranose-(1-3)-alpha-L-arabinofuranose-(1-3)-alpha-D-mannopyranose-(1-2)-[alpha-L-arabinofuranose-(1-2)-alpha-L-arabinofuranose-(1-4)][alpha-L-arabinofuranose-(1-6)]beta-D-galactopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]alpha-D-mannopyranose-(1-4)-alpha-D-mannopyranose-(1-4)-[alpha-L-arabinofuranose-(1-4)-alpha-D-mannopyranose-(1-3)-[alpha-L-arabinofuranose-(1-3)-[alpha-L-arabinofuranose-(1-6)]beta-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-[alpha-L-arabinofuranose-(1-2)][alpha-L-arabinofuranose-(1-6)]beta-D-mannopyranose-(1-6)][beta-L-arabinofuranose-(1-2)]alpha-D-mannopyranose-(1-6)-[alpha-L-arabinofuranose-(1-3)]2-acetamido-2-deoxy-beta-D-galactopyranose-(1-4)-[alpha-L-arabinofuranose-(1-3)-beta-L-arabinofuranose-(1-2)]beta-D-mannopyranose-(1-4)-beta-D-mannopyranose-(1-2)-[alpha-L-arabinofuranose-(1-5)]beta-L-arabinofuranose


Type: oligosaccharide / Mass: 4280.733 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LArafa1-3LArafa1-3DManpa1-2[LArafa1-2LArafa1-4][LArafa1-6]DGalpb1-3[DManpa1-6]DManpa1-4DManpa1-4[LArafa1-4DManpa1-3[LArafa1-3[LArafa1-6]DManpb1-6]DManpb1-4[LArafa1-2][LArafa1-6]DManpb1-6][LArafb1-2]DManpa1-6[LArafa1-3]DGalpNAcb1-4[LArafa1-3LArafb1-2]DManpb1-4DManpb1-2[LArafa1-5]LArafb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/6,29,28/[a211h-1b_1-4][a1122h-1b_1-5][a211h-1a_1-4][a2112h-1b_1-5_2*NCC/3=O][a1122h-1a_1-5][a2112h-1b_1-5]/1-2-2-1-3-4-3-5-1-5-5-6-5-3-3-3-3-3-5-2-3-2-5-3-2-3-3-3-3/a2-b1_a5-C1_b4-c1_c2-d1_c4-f1_d3-e1_f3-g1_f6-h1_h2-i1_h4-j1_h6-t1_j4-k1_k3-l1_k6-s1_l2-m1_l4-p1_l6-r1_m3-n1_n3-o1_p2-q1_t2-u1_t4-v1_t6-B1_v3-w1_v6-y1_w4-x1_y3-z1_y6-A1WURCSPDB2Glycan 1.1.0
[][L-1-deoxy-Araf]{[(2+1)][b-D-Manp]{[(4+1)][b-D-Manp]{[(2+1)][b-L-Araf]{[(3+1)][a-L-Araf]{}}[(4+1)][b-D-GalpNAc]{[(3+1)][a-L-Araf]{}[(6+1)][a-D-Manp]{[(2+1)][b-L-Araf]{}[(4+1)][a-D-Manp]{[(4+1)][a-D-Manp]{[(3+1)][b-D-Galp]{[(2+1)][a-D-Manp]{[(3+1)][a-L-Araf]{[(3+1)][a-L-Araf]{}}}[(4+1)][a-L-Araf]{[(2+1)][a-L-Araf]{}}[(6+1)][a-L-Araf]{}}[(6+1)][a-D-Manp]{}}}[(6+1)][b-D-Manp]{[(2+1)][a-L-Araf]{}[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{[(4+1)][a-L-Araf]{}}[(6+1)][b-D-Manp]{[(3+1)][a-L-Araf]{}[(6+1)][a-L-Araf]{}}}[(6+1)][a-L-Araf]{}}}}}}[(5+1)][a-L-Araf]{}}LINUCSPDB-CARE
#4: Polysaccharide
alpha-L-arabinofuranose-(1-2)-alpha-L-arabinofuranose-(1-3)-alpha-L-arabinofuranose-(1-2)-alpha-D- ...alpha-L-arabinofuranose-(1-2)-alpha-L-arabinofuranose-(1-3)-alpha-L-arabinofuranose-(1-2)-alpha-D-mannopyranose-(1-2)-[beta-L-arabinofuranose-(1-2)-alpha-L-arabinofuranose-(1-3)-alpha-L-arabinofuranose-(1-3)]alpha-L-arabinofuranose-(1-4)-[alpha-D-mannopyranose-(1-2)-alpha-D-mannopyranose-(1-2)-alpha-L-arabinofuranose-(1-2)]beta-D-glucopyranose-(1-4)-[alpha-D-mannopyranose-(1-2)]beta-D-mannopyranose-(1-4)-[alpha-L-arabinofuranose-(1-2)]beta-D-galactopyranose


Type: oligosaccharide / Mass: 2342.034 Da / Num. of mol.: 18
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LArafa1-2LArafa1-3LArafa1-2DManpa1-2[LArafb1-2LArafa1-3LArafa1-3]LArafa1-4[DManpa1-2DManpa1-2LArafa1-2]DGlcpb1-4[DManpa1-2]DManpb1-4[LArafa1-2]DGalpb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/6,16,15/[a2112h-1b_1-5][a211h-1a_1-4][a1122h-1b_1-5][a1122h-1a_1-5][a2122h-1b_1-5][a211h-1b_1-4]/1-2-3-4-5-2-4-4-2-4-2-2-2-2-2-6/a2-b1_a4-c1_c2-d1_c4-e1_e2-f1_e4-i1_f2-g1_g2-h1_i2-j1_i3-n1_j2-k1_k3-l1_l2-m1_n3-o1_o2-p1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-Galp]{[(2+1)][a-L-Araf]{}[(4+1)][b-D-Manp]{[(2+1)][a-D-Manp]{}[(4+1)][b-D-Glcp]{[(2+1)][a-L-Araf]{[(2+1)][a-D-Manp]{[(2+1)][a-D-Manp]{}}}[(4+1)][a-L-Araf]{[(2+1)][a-D-Manp]{[(2+1)][a-L-Araf]{[(3+1)][a-L-Araf]{[(2+1)][a-L-Araf]{}}}}[(3+1)][a-L-Araf]{[(3+1)][a-L-Araf]{[(2+1)][b-L-Araf]{}}}}}}}LINUCSPDB-CARE
Has ligand of interestY
Has protein modificationY

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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: TLP-4 / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: algae metagenome (others)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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: DIFFRACTION / Nominal defocus max: 1800 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -134.79 ° / Axial rise/subunit: 12.41 Å / Axial symmetry: C1
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 27189 / Symmetry type: HELICAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049772
ELECTRON MICROSCOPYf_angle_d1.17813260
ELECTRON MICROSCOPYf_dihedral_angle_d14.2885470
ELECTRON MICROSCOPYf_chiral_restr0.0623348
ELECTRON MICROSCOPYf_plane_restr0.00188

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