ジャーナル: J Virol / 年: 2013 タイトル: Influences on trimerization and aggregation of soluble, cleaved HIV-1 SOSIP envelope glycoprotein. 著者: Per Johan Klasse / Rafael S Depetris / Robert Pejchal / Jean-Philippe Julien / Reza Khayat / Jeong Hyun Lee / Andre J Marozsan / Albert Cupo / Nicolette Cocco / Jacob Korzun / Anila Yasmeen / ...著者: Per Johan Klasse / Rafael S Depetris / Robert Pejchal / Jean-Philippe Julien / Reza Khayat / Jeong Hyun Lee / Andre J Marozsan / Albert Cupo / Nicolette Cocco / Jacob Korzun / Anila Yasmeen / Andrew B Ward / Ian A Wilson / Rogier W Sanders / John P Moore / 要旨: We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as ...We describe methods to improve the properties of soluble, cleaved gp140 trimers of the human immunodeficiency virus type 1 (HIV-1) envelope glycoproteins (Env) for use in structural studies and as immunogens. In the absence of nonionic detergents, gp140 of the KNH1144 genotype, terminating at residue 681 in gp41 (SOSIP.681), has a tendency to form higher-order complexes or aggregates, which is particularly undesirable for structure-based research. We found that this aggregation in the absence of detergent does not involve the V1, V2, or V3 variable regions of gp120. Moreover, we observed that detergent forms micelles around the membrane-proximal external region (MPER) of the SOSIP.681 gp140 trimers, whereas deletion of most of the MPER residues by terminating the gp140 at residue 664 (SOSIP.664) prevented the aggregation that otherwise occurs in SOSIP.681 in the absence of detergent. Although the MPER can contribute to trimer formation, truncation of most of it only modestly reduced trimerization and lacked global adverse effects on antigenicity. Thus, the MPER deletion minimally influenced the kinetics of the binding of soluble CD4 and a CD4-binding site antibody to immobilized trimers, as detected by surface plasmon resonance. Furthermore, the MPER deletion did not alter the overall three-dimensional structure of the trimers, as viewed by negative-stain electron microscopy. Homogeneous and aggregate-free MPER-truncated SOSIP Env trimers are therefore useful for immunogenicity and structural studies.
ダウンロード / ファイル: emd_5706.map.gz / 形式: CCP4 / 大きさ: 15.3 MB / タイプ: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
注釈
Negative stained image reconstruction of HIV KNH11444 subtype A SOSIP.664 in complex with Fab fragment of VRC PG04 monoclonal antibody
ボクセルのサイズ
X=Y=Z: 2.18 Å
密度
表面レベル
登録者による: 3.21 / ムービー #1: 3.21
最小 - 最大
-5.97782993 - 17.474040989999999
平均 (標準偏差)
0.0 (±1.0)
対称性
空間群: 1
詳細
EMDB XML:
マップ形状
Axis order
X
Y
Z
Origin
0
0
0
サイズ
160
160
160
Spacing
160
160
160
セル
A=B=C: 348.80002 Å α=β=γ: 90.0 °
CCP4マップ ヘッダ情報:
mode
Image stored as Reals
Å/pix. X/Y/Z
2.18
2.18
2.18
M x/y/z
160
160
160
origin x/y/z
0.000
0.000
0.000
length x/y/z
348.800
348.800
348.800
α/β/γ
90.000
90.000
90.000
start NX/NY/NZ
-132
-122
-147
NX/NY/NZ
250
274
261
MAP C/R/S
1
2
3
start NC/NR/NS
0
0
0
NC/NR/NS
160
160
160
D min/max/mean
-5.978
17.474
-0.000
-
添付データ
-
試料の構成要素
-
全体 : HIV KNH11444 subtype A SOSIP.664 in complex with Fab fragment of ...
全体
名称: HIV KNH11444 subtype A SOSIP.664 in complex with Fab fragment of VRC PG04 monoclonal antibody
要素
試料: HIV KNH11444 subtype A SOSIP.664 in complex with Fab fragment of VRC PG04 monoclonal antibody
タンパク質・ペプチド: Human immunodeficiency virus Envelope protein
タンパク質・ペプチド: Fab fragment of VRC PG04 monoclonal antibody
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超分子 #1000: HIV KNH11444 subtype A SOSIP.664 in complex with Fab fragment of ...
超分子
名称: HIV KNH11444 subtype A SOSIP.664 in complex with Fab fragment of VRC PG04 monoclonal antibody タイプ: sample / ID: 1000 / 詳細: The sample was monodisperse via SEC. / 集合状態: Trimer / Number unique components: 2
分子量
理論値: 613 KDa
-
分子 #1: Human immunodeficiency virus Envelope protein
試料ホルダーモデル: SIDE ENTRY, EUCENTRIC / Tilt angle max: 55
実験機器
モデル: Tecnai F20 / 画像提供: FEI Company
+
画像解析
詳細
Particles were selected using DogPicker, reference free class averages determined using Sparx, and initial model generated using common-lines with EMAN2.
CTF補正
詳細: Each particle
最終 再構成
アルゴリズム: OTHER / 解像度のタイプ: BY AUTHOR / 解像度: 15.0 Å / 解像度の算出法: FSC 0.5 CUT-OFF / ソフトウェア - 名称: Sparx/EMAN2 / 詳細: Final maps were calculated from one data set. / 使用した粒子像数: 12236