PDBsum entry 3inu

Immune system

PDB id: 3inu

Contents

Protein chains

226 a.a. *

217 a.a. *

Ligands

SO4 ×8

GOL ×4

Waters ×76

* Residue conservation analysis

PDB id:

3inu

Name:

Immune system

Title:

Crystal structure of an unbound kz52 neutralizing anti-ebolavirus antibody.

Structure:

Kz52 antibody fragment heavy chain. Chain: h, m. Engineered: yes. Kz52 antibody fragment light chain. Chain: l, n. Engineered: yes

Source:

Homo sapiens. Organism_taxid: 9606. Expressed in: cricetulus griseus. Expression_system_taxid: 10029. Expression_system_taxid: 10029

Resolution:

2.50Å R-factor: 0.205 R-free: 0.252

Authors:

J.E.Lee,M.L.Fusco,D.M.Abelson,A.J.Hessell,D.R.Burton,E.O.Saphire

Key ref:

J.E.Lee et al. (2009). Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein. Acta Crystallogr D Biol Crystallogr, 65, 1162-1180. PubMed id: 19923712

Date:

12-Aug-09 Release date: 27-Oct-09

Protein chains

No UniProt id for this chain

Struc: 226 a.a.

Protein chains

No UniProt id for this chain

Struc: 217 a.a.

Acta Crystallogr D Biol Crystallogr 65:1162-1180 (2009)

PubMed id: 19923712

Techniques and tactics used in determining the structure of the trimeric ebolavirus glycoprotein.

J.E.Lee, M.L.Fusco, D.M.Abelson, A.J.Hessell, D.R.Burton, E.O.Saphire.

ABSTRACT

The trimeric membrane-anchored ebolavirus envelope glycoprotein (GP) is responsible for viral attachment, fusion and entry. Knowledge of its structure is important both for understanding ebolavirus entry and for the development of medical interventions. Crystal structures of viral glycoproteins, especially those in their metastable prefusion oligomeric states, can be difficult to achieve given the challenges in production, purification, crystallization and diffraction that are inherent in the heavily glycosylated flexible nature of these types of proteins. The crystal structure of ebolavirus GP in its trimeric prefusion conformation in complex with a human antibody derived from a survivor of the 1995 Kikwit outbreak has now been determined [Lee et al. (2008), Nature (London), 454, 177-182]. Here, the techniques, tactics and strategies used to overcome a series of technical roadblocks in crystallization and phasing are described. Glycoproteins were produced in human embryonic kidney 293T cells, which allowed rapid screening of constructs and expression of protein in milligram quantities. Complexes of GP with an antibody fragment (Fab) promoted crystallization and a series of deglycosylation strategies, including sugar mutants, enzymatic deglycosylation, insect-cell expression and glycan anabolic pathway inhibitors, were attempted to improve the weakly diffracting glycoprotein crystals. The signal-to-noise ratio of the search model for molecular replacement was improved by determining the structure of the uncomplexed Fab. Phase combination with Fab model phases and a selenium anomalous signal, followed by NCS-averaged density modification, resulted in a clear interpretable electron-density map. Model building was assisted by the use of B-value-sharpened electron-density maps and the proper sequence register was confirmed by building alternate sequences using N-linked glycan sites as anchors and secondary-structural predictions.