著者らは、DNA精製のヘルプについては、データ収集とモニカPillon時の援助のためのNSLS（ブルックヘブン国立研究所）でPXRRのスタッフに感謝したいと思います。この作品は、カナダ衛生研究所（MOP 67189）によってサポートされていました。

1。タンパク質精製接続するフレキシブルリンカー、N-（オリゴマー）およびC末端（DNA結合）SeqAエイズ、DNA上の一から三ターンで区切らヘミメチルGATCリピートの認識のドメイン。この研究では、我々はさらに、オリゴマーとによって排他的に分離されたタンデムGATCへのDNA結合繰り返しを制限短縮しリンカを防ぎN末端ドメインに点突然変異とSeqAの二量体変異体（SeqAΔ（41から59）-A25R）を使用DNA上の1ターン（ 図1）2,8。 <ol><li> T7プロモーターの制御下にコードするプラスミドSeqAとBL21（DE3）細胞を形質転換し、 </li><li> 100μg/ mlのアンピシリンを含むLB寒天プレートにプレート変換反応、 </li><li>小規模の一晩培養（100μg/ mlのアンピシリンを含むLB培地）に接種し混合コロニーをピックアップ、 </li><li>翌朝overniの1:100希釈液を使用したメディアの1リットルに接種GHT文化、 </li><li> 〜0.7のOD 600に細胞を成長し、終濃度1mMにイソプロピルβ-D-1-チオガラクトピラノシド（IPTG）を添加することにより、タンパク質産生を誘導し、 </li><li> 、軌道に振とうしながら37℃で3時間インキュベーションを継続した後、遠心分離（3,300×gで10分）により細胞を収穫</li><li>精製バッファーで細胞ペレットを再懸濁し、超音波処理により溶解、 </li><li>遠心分離（39,000×gで40分間）によって溶解物をクリアして、精製バッファーで平衡化したヘパリンカラムに上清をロードし、 </li><li> 1MのNaCl（〜0.7MのNaClで溶出SeqA）の直線勾配を用いて溶出SeqA、 </li><li>一緒にプールSeqA含有画分を精製バッファーで平衡化した陽イオン交換クロマトグラフィーカラムに試料と負荷のイオン強度を下げるために希釈し、 </li><li>線形塩勾配を用いて、純粋SeqAは、〜0.4 MのNaClで溶出</li><li>一緒にプールSeqA含有画分を、concentratE（3 mg / ml）とストレージ·バッファに保管してください。 </li></ol> 2。 DNA精製<ol><li>あなたの好みの会社から順に相補非メチル化及びメチル化オリゴヌクレオチド、 </li><li> 、オートクレーブ滅菌ddH 2 Oを、渦の800μlの各凍結乾燥した一本鎖DNAの1マイクロモルを溶解し、それが10〜20分間放置</li><li>各オリゴヌクレオチドに予熱2Xローディングバッファー800μlを添加し、 </li><li> 20から30ヌクレオチド長のオリゴヌクレオチドは、大規模な10パーセント変性ゲル（160×250×1mm）を準備します。 *よく10％ページミックス、80μlのTEMEDとゲルあたり800μlの過硫酸アンモニウムの80ミリリットルを混合し、注ぐ、 一旦重合*、コームを取り外し、徹底的にddH 2 Oで井戸をすすぎ、 *、冷却板を含むゲルキャスト上でゲルを組み立て、その後バッファー（1×TBE）を実行して上部と下部貯水池を埋める * 55℃にゲルを暖めるために700から750 Vでゲルを事前実行、 *実行を停止し、井戸ソロをすすぐughlyバッファが実行されています。 </li><li> 90にオリゴヌクレオチドを​​熱し℃で2分間、 </li><li>渦とサンプルをスピンし、すぐにゲルをロードする前に、 </li><li> 〜700 Vでゲルを実行して、オリゴヌクレオチドが中途半端に移行した後、停止します。 （10％ポリアクリルアミドゲル上でブロモフェノールブルーのオリゴヌクレオチドとの共同移行していることに注意してください〜〜60塩基の長さを持つ20塩基長とキシレンシアノールFF）を、 </li><li>ゲルを停止し、ゲルボックスからそれを分解してスペーサーを取り外す</li><li>平らな面に、1枚のガラスプレートを取り外し、ラップでゲルを覆い、 </li><li> 、、ゲルを好転他のガラス板を取り外して、プラスチック製のラップでそれをカバー</li><li> 、DNAの影を見てゲルの後ろに紫外光や蛍光プレートを用いてバンドをマーク</li><li>小さい部分にカミソリの刃でバンドを切り取り、無菌の15mlチューブにそれらを転送し、 </li><li>溶出緩衝液9mlを追加し、攪拌しながら37℃で一晩溶出、 </li><li>慎重に溶液を移す転送アクリルアミドの部分を避けて、pH7の3 M酢酸ナトリウム（1:10希釈）を1ml加えて冷やした100％エタノール（2.5巻）の25ミリリットルを追加するには、ゲルローディングピペットチップを用いてオートクレーブした遠心管に、 </li><li>少なくとも3時間-20℃でインキュベートし、 </li><li> 、スピンダウンし、別のチューブに上清を移す</li><li>中火速度-VACでペレットを乾燥させ、 </li><li>オートクレーブ滅菌ddH 2 Oを400μlにペレットを再懸濁し、新しいチューブに移し、 </li><li> 100％エタノールのpHは7と1ミリリットルで3 M酢酸ナトリウム40μlを加え、よく混ぜる（渦）、-20℃で30分間、続いて室温で30分間℃で、インキュベート</li><li> 、18,000 gで15分間スピンし、上清を捨てる</li><li> 18000グラムで6分間ペレットとスピンから残留塩を除去するために70パーセントの冷エタノールを100μlでペレットを洗浄します。 、エタノールを捨て、スピード-VACでペレットを乾燥させ</li><li>オートクレーブ滅菌蒸留H100μlの合計でペレットを再懸濁し<sub&gt; 2 O 、オリゴヌクレオチドの濃度を測定</li><li>ヘミメチル化DNA二重鎖をアニーリングするように、彼らが水浴内部ゆっくりと室温まで冷却させた後相補的な一本鎖のモル濃度をミックスし、℃で5分間水浴中で95に混合物を加熱して。 </li></ol> 3。タンパク質-DNA複合体形成と分析<ol><li>精製SeqAΔ等量の（41から59）-A25（81μM）およびヘミメチル化DNA（81μM）を、ミックス</li><li>あなたはそれを使用する準備が整うまでは、4時15分、店舗℃の室温でインキュベート</li><li>商用スパースマトリックス画面を使用して結晶化条件の画面、 </li><li>一度初期結晶化のリード線は回折品質結晶を成長させるための条件を最適化し、同定されており、 </li><li>どちらか25％の最終濃度（に結晶化溶液にPEG400の存在量を増加させることによって得られたSeqA-DNAの結晶をCryoprotectv / v）で、または、結晶化溶液に20％グリセロール（v / v）を追加</li><li>ナイロンループを使って個々の結晶をすくい、液体窒素中でそれらをフラッシュフリーズ、 </li><li> 100 Kの各結晶の回折限界をテスト</li></ol> 4。代表的な結果ヘミメチルGATC配列間の分離（i）は、二重の（ii）の長さ、および（iii）：ヘミメチル化DNAに結合したSeqAΔ（41から59）-A25Rの結晶構造を得るために、我々は連続してDNA上の3つのパラメータを最適化5 &#39;オーバーハングの不在/存在。 エレクトロモビリティシフトアッセイはSeqAΔ（41から59）-A25Rが優先的に9から10塩基対（ 図1）で区切られたGATC繰り返しを結合することを示す。したがって、我々は最初に長い9または10 bpsのどちらかで区切られた二つのヘミメチルGATC配列を含む（bps）の二重鎖23から24塩基対をスクリーニングした。三二本鎖は、うまく形の結晶が（ 図2）を得た。アル高分解能で回折結晶のしかしなし、9 bpsのGATC分離、結晶化のために好まれたことを示す他の部分よりもX線回折より良い9 bpsには、で区切られた二つのGATCサイトと23 bpsの長い二重。したがって、我々は後続のすべての画面に9 bpsに間GATC間隔を修正しました。 複数のDNA分子が結晶内で9連続B型DNAを形成するために、頭部と尾部を積み重ねることができるので、一般的には、DNAの結晶化は、正確なヘリカル巻数に対応する二重の長さについては好まれています。したがって、我々は21 bpsの（ すなわち、2つのらせん状の回転）に二重鎖の全長を短縮した。平滑末端を有する21 bpsの全二重では回折品質結晶（データは示さず）、結晶を得たそれぞれの端に単一ヌクレオチドの5 &#39;オーバーハングを有する21 bpsの半二重その私達の家のソース内の5Åの回折。を得ませんでしたが回折限界の改善は、エンドツーエンドのデュプレックス協会は確かに有利であることが示唆されたる結晶のパッキング。 SeqAがDNAの同一面上にあるGATC配列と相互作用するので、DNA二重鎖の反対側の面は、溶剤にさらされるべきである。さらに複合体の結晶性を向上するために、我々は、メソッド、二重の反対側の面を通って結晶に隣接したDNA分子を用いた溝バックボーン相互作用を促進するために2つのサイト間のGATC CGジヌクレオチドを​​含むように二重の順序を変更それは、過去10のDNAの結晶化を強化するために使用されています。しかし、CGを含有二重で成長した結晶の回折限界はCGジヌクレオチド（ 図3）を含んでいなかった似たような二本鎖DNAとの相互成長したものと同一であった。この結果は、溝のバックボーン相互作用がこのケースでは重要ではないことが示された。我々は、その後それぞれに2付加的なヌクレオチドを​​持っていた二本鎖DNAと成長した結晶を比較することにより、オーバーハングの長さを最適化5 &#39;末端。この変化は、結晶形態の抜本的な効果を持っていたと同様に、回折限界は、追加のヌクレオチドが劇的に強化された分子の連絡先や結晶組織（ 図3）を変更したことを示す。 SeqAΔ（41から59）-A25Rの結晶構造は、DNA二重鎖の自由面が結晶接触に従事しないことを確認し、この最後の二本鎖DNAに結合した、など、CG-ジヌクレオチドを​​導入する効果は限定的と予想。タンパク質とDNAの相対的な大きさにもかかわらず、対称の仲間の間でほとんどの相互作用は、タンパク質-タンパク質とタンパク質-DNA相互作用（ 図4）によって媒介される。興味深いことに、この特定のケースでは、5 &#39;オーバーハングヌクレオチドの有益な効果は、擬似連続DNAの形成によるものではない。その代わりに、離れたDNA軸からのメチル化鎖プロジェクトの5 &#39;末端とその理由2ヌクレオチドワット説明して、複合体の近位SeqA分子と相互作用する<html> EREは厳密には8,11のパッキング結晶を変更する必要がありました。 <img alt="図1" src="/files/ftp_upload/4266/4266fig1.jpg" /> 図1。ヘミメチル化DNAへSeqAΔ（41から59）-A25Rの結合。 （A）が媒介するタンパク質のオリゴマー化とDNA結合そのSeqAのドメインの概略図を。塩基対の増加によって分離された二つのヘミメチルGATC配列を含むDNAとSeqAΔ（41から59）-A25R（B）の電気泳動移動度シフトアッセイ（ X）である。一番左のレーンは（空白のラベルが付いている）SeqAΔ（41から59）-A25Rの非存在下での2 GATC配列間の5,7を有するDNA、12、21、25〜34塩基対の等モル混合物を含んでいます（C）図は、回折品質結晶を得るためにDNA二本鎖に最適化された三つの変数を描いた。 upload/4266/4266fig2.jpg &quot;のfo：コンテンツの幅=&quot; 6インチ &quot;のfo：SRC =&quot; / files/ftp_upload/4266/4266fig2highres.jpg &quot;/&gt; 図2。 9または10塩基対で区切られた二つのヘミメチルGATC部位を含む23から24 bpの長さの二本鎖を用いて得られた使用したオリゴヌクレオチドと結晶の間GATC距離。概要を変化させる効果 。結晶のすべての画像が同じ倍率で撮影し、スケールバーは100μmを示した。各SeqAΔ（41から59） - A25R-DNAの結晶の分解能限界はリガクのRU-300のX線発生装置のシステムで収集された回折像に基づいています。 <a href="http://www.jove.com/files/ftp_upload/4266/4266fig2large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img alt="図3" src="/files/ftp_upload/4266/4266fig3.jpg" fo:content-width="6in" fo:src="/files/ftp_upload/4266/4266fig3highres.jpg" /> 図3。トンのDNAを変化させる効果が終了します。まとめ21 bpsの長い二本鎖が9塩基対で区切られた二つのヘミメチルGATC部位を含む、0、1、または2ヌクレオチド5 &#39;末端オーバーハングを含むと彼オリゴヌクレオチドが使用され、結晶が得られた。結晶のすべての画像が同じ倍率で撮影し、スケールバーは100μmを示した。各SeqAΔ（41から59） - A25R-DNAの結晶の分解能限界はX12CとX29（NSLS、BNL）のビームラインで収集された回折像に基づいています。 <a href="http://www.jove.com/files/ftp_upload/4266/4266fig3large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img alt="図4" src="/files/ftp_upload/4266/4266fig4.jpg" /> 図4。ジヌクレオチドオーバーハングとDNAに結合SeqAΔ（41から59）-A25Rの結晶充填：（）上面と側面（B）から見て。非対称単位は、DNAがオレンジ色で表示されている間タンパク質がグレーで表示されます2SeqAΔ（41から59） - A25R-DNA複合体を含んでいます。対称RY関連SeqAΔ（41から59） - A25R-DNA分子は、タンパク質とDNAのために黄色に白で表示されます。この数字はpymolの12を使用して調製した。この数字は、 ムービー1に関連しています。 <a href="http://www.jove.com/files/ftp_upload/4266/4266fig4large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 映画は1。 <a href="http://www.jove.com/files/ftp_upload/4266/4266movie1.avi" target="_blank">ムービーを見るにはここをクリック</a> 。

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特別な利害関係は宣言されません。

高分子のX線結晶学における最大の課題の1つは、回折品質の結晶を得ることである。タンパク質またはタンパク質-DNA複合体の場合には、このチャレンジが最適化されなければならない追加の変数が原因で悪化している。それは広く利用されているDNAの長さと粘り気のオーバーハングの存在は長く擬似二重に隣接するDNA分子の関連付けを強化すると考えられている最適化するための主要なパラ?...

<table border="1"><tbody><tr><td> 試薬の名称 </td><td> 会社 </td><td> カタログ＃ </td><td> コメント（オプション） </td></tr><tr><td>トリス</td><td> Bioshop </td><td> TRS003.5 </td><td></td></tr><tr><td>エチレンジアミン四酢酸（EDTA） </td><td>フィッシャー·サイエンティフィック</td><td> E478-500 </td><td></td></tr><tr><td> Dithiotheitrolトール（DTT） </td><td>基本バイオ株式会社</td><td> DB0058 </td><td></td></tr><tr><td> NaClを</td><td> Bioshop </td><td> SOD002.10 </td><td></td></tr><tr><td>グリセロール</td><td>カレドン</td><td> 5350から1 </td><td></td></tr><tr><td>スクロース</td><td>シグマアルドリッチ</td><td> S5016-500G </td><td></td></tr><tr><td>ドデシル硫酸ナトリウム（SDS） </td><td> Bioshop </td><td> SDS001.500 </td><td></td></tr><tr><td>尿素</td><td> Bioshop </td><td> URE001.5 </td><td></td></tr><tr><td> 40パーセント29:1ビス/アクリルアミド</td><td>基本バイオ株式会社</td><td> A0007-500ML </td><td> 4℃で保存</td></tr><tr><td>ホウ酸</td><td> EMD </td><td> BX0865-1 </td><td></td></tr><tr><td>キシレンシアノールFF </td><td>バイオ·ラッド</td><td> 161-0423 </td><td></td></tr><tr><td>ブロモフェノールブルー</td><td> Bioshop </td><td> BR0222 </td><td></td></tr><tr><td>デュアル可変垂直ゲルシステム</td><td> CBCのサイエンティフィック·カンパニー·インク</td><td> DASG-250 </td><td></td></tr><tr><td>インデックス晶画面</td><td>ハンプトンリサーチ</td><td> HR2-144 </td><td> 4℃で保存</td></tr><tr><td>ウィザード私は結晶化画面</td><td>エメラルドアプライドバイオシステムズ</td><td> EBS-WIZ-1 </td><td> 4℃で保存</td></tr><tr><td>ウィザードIIの結晶化画面</td><td&gt;エメラルドアプライドバイオシステムズ</td><td> EBS-WIZ-2 </td><td> 4℃で保存</td></tr><tr><td>クラシック結晶化画面</td><td>キアゲン</td><td> 130701 </td><td> 4℃で保存</td></tr><tr><td> Intelliplateトレー</td><td>アートロビンスインスツルメンツ</td><td> 102-0001-00 </td><td></td></tr><tr><td></td><td></td><td></td><td>ソリューション タンパク質精製バッファー：100 mMトリスpH 8、2mMのEDTA、2mMのDTT、5％グリセロール。 タンパク質貯蔵バッファー：20 mMトリスpH 8、150mMのNaCl、5mMのDTT、0.5mMのEDTA、5％グリセロール。 ゲルローディングミックス：オートクレーブ滅菌蒸留H 2 Oの70ミリリットルにショ糖の20グラム、ブロモフェノールブルーを25mg、キシレンシアノールFFの25mg、10％（w / v）のSDSおよび10X TBEの10ミリリットルの1ミリリットルを追加ショ糖が溶解するまで穏やかに加熱して攪拌し、最終的な調整オートクレーブ滅菌蒸留H 2 Oで100mlにボリューム4℃で保存する 2Xローディングバッファー：ゲルローディングミックス10mlに尿素の11グラムを追加します。尿素が溶けるまで、ホットプレート上でかき混ぜる。 4で2 mlチューブ内や店舗内にアリコート℃、 10Xホームページミックス：ミックス尿素の420.4グラム、10X TBEの100ミリリットル（オートクレーブ）、40％、250mlの29:1のddH 2 Oのビス/アクリルアミド完全に溶解するまで攪拌し、1リットルにボリュームを調整します。 4℃の暗所に保管してボトル 10X TBE：ディゾルブ 108グラム トリス、蒸留H 2 Oの1リットル中のホウ酸の55グラムとEDTAの9.3グラムの室温でオートクレーブや店舗。 溶出緩衝液：蒸留H 2 Oの200ミリリットルの5 M NaClを、1Mトリス（pH7.5）、0.5MのEDTA pHが8の0.4ミリリットルの2ミリリットルの希薄8ミリリットル室温でオートクレーブや店舗。 </td></tr></tbody></tabLE&gt;

iterative optimization of dna duplexes for crystallization of seqa-dna complexes

大腸菌 SeqAは複製1の原点内封鎖ヘミメチルGATCクラスターによる早期再開始イベントを防ぐDNA複製の負の調節因子である。原点を超えて、SeqAは複製フォーク、それがより高い秩序構造に新しく複製されたDNAを整理2で見出される。 SeqAシングルGATC配列と共に弱くしか仲間が、それは複数GATC部位を含むDNA二重鎖と親和性の高い複合体を形成している。 SeqAの最小限の機能と構造単位は、それによってヘミメチル化DNA 3と高親和性複合体を形成するために少なくとも2つのGATCシーケンスの要件を説明し、二量体である。さらに、SeqAアーキテクチャは、オリゴマー化と柔軟なリンカーによって分離されたDNA結合ドメインと、3つの螺旋回転までで区切らGATCリピートに結合することができます。したがって、分子レベルでSeqAの機能を理解することは構造的アナログを必要と複数GATC配列に結合SeqAの溶解。タンパク質-DNAの結晶化では、DNAはタンパク質とDNAの相対サイズとアーキテクチャに応じてパッキング相互作用に非常に優れた効果をnoneに持つことはできません。タンパク質はDNAまたはDNAの足跡のほとんどよりも大きい場合には、結晶充填は、主に、タンパク質 - タンパク質相互作用によって媒介される。逆に、タンパク質はDNAよりも同じサイズ以下であるか、またはそれが唯一のDNA、DNA-DNAとDNA-タンパク質相互作用の一部をカバーするとき、結晶パッキングを支配している。したがって、タンパク質-DNA複合体の結晶化は、DNAの長さ4、DNA末端（鈍またはオーバーハング）5-7の体系的スクリーニングを必要とします。本稿では、設計、最適化、精製および構造決定に適した結晶を得ることがSeqAの二量体変異体（SeqAΔ（41から59）-A25R）と複合したタンデムGATCリピートを含むヘミメチル化DNA二重鎖を結晶化する方法について説明します。

Watch this Scientific Journal Video about Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes at JoVE.com

Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

タンパク質-DNA複合体の結晶構造は、タンパク質の機能、機構、ならびに、特定の相互作用の性質についての洞察を提供することができます。ここでは、との共結晶の長さ、配列および二本鎖DNAの末端を最適化する方法を報告して<em&gt;大腸菌</em&gt; SeqA、複製開始の負の調節因子。

SeqA-DNA複合体の結晶化のためのDNA二重鎖の反復最適化

Escherichia coli SeqA is a negative regulator of DNA replication that prevents premature reinitiation events by sequestering hemimethylated GATC clusters ...

iterative-optimization-dna-duplexes-for-crystallization-seqa-dna

Research

JoVE Journal

Biology

9.9K ビュー.  McMaster University. タンパク質-DNA複合体の結晶構造は、タンパク質の機能、機構、ならびに、特定の相互作用の性質についての洞察を提供することができます。ここでは、との共結晶の長さ、配列および二本鎖DNAの末端を最適化する方法を報告して大腸菌 SeqA、複製開始の負の調節因子。

ビデオ: Iterative Optimization of DNA Duplexes for Crystallization of SeqA-DNA Complexes

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

電子線結晶学による構造生物学研究用小型膜タンパク質の二次元結晶化トライアルの評価

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

生物学

Protein Crystallization for X-ray Crystallography

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The availability of atomic resolution structures provides a deep and unique understanding of protein function, and helps to unravel the inner workings of the living cell. To date, 86% of the Protein Data Bank (rcsb-PDB) entries are macromolecular structures that were determined using X-ray crystallography.
To obtain crystals suitable for crystallographic studies, the macromolecule (e.g. protein, nucleic acid, protein-protein complex or protein-nucleic acid complex) must be purified to homogeneity, or as close as possible to homogeneity. The homogeneity of the preparation is a key factor in obtaining crystals that diffract to high resolution (Bergfors, 1999; McPherson, 1999).
Crystallization requires bringing the macromolecule to supersaturation. The sample should therefore be concentrated to the highest possible concentration without causing aggregation or precipitation of the macromolecule (usually 2-50 mg/ mL). Introducing the sample to precipitating agent can promote the nucleation of protein crystals in the solution, which can result in large three-dimensional crystals growing from the solution. There are two main techniques to obtain crystals: vapor diffusion and batch crystallization. In vapor diffusion, a drop containing a mixture of precipitant and protein solutions is sealed in a chamber with pure precipitant. Water vapor then diffuses out of the drop until the osmolarity of the drop and the precipitant are equal (Figure 1A). The dehydration of the drop causes a slow concentration of both protein and precipitant until equilibrium is achieved, ideally in the crystal nucleation zone of the phase diagram. The batch method relies on bringing the protein directly into the nucleation zone by mixing protein with the appropriate amount of precipitant (Figure 1B). This method is usually performed under a paraffin/mineral oil mixture to prevent the diffusion of water out of the drop.
Here we will demonstrate two kinds of experimental setup for vapor diffusion, hanging drop and sitting drop, in addition to batch crystallization under oil.

The 3-D structure of a molecule provides a unique understanding of how the molecule functions. The principal method for structure determination at near-atomic resolution is X-ray crystallography. Here, we demonstrate the current methods for obtaining three-dimensional crystals of any given macromolecule that are suitable for structure determination by X-ray crystallography.

X -線結晶構造のためのタンパク質の結晶化

Using the three-dimensional structure of biological macromolecules to infer how they function is one of the most important fields of modern biology. The ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

脂質中間相における膜タンパク質の結晶化

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

脂質Bicelle法を用いた膜タンパク質のハイスループット結晶化

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

脂質中間相における膜タンパク質のハイスループット結晶化のためのロボットの使用

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

植物タンパク質複合体の翻訳後修飾の同定

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short period of time. However, this technology is only just beginning to be applied to large-scale production as many technological obstacles to scale up are now being overcome. Here, we demonstrate a simple and reproducible method for industrial-scale transient protein production based on vacuum infiltration of Nicotiana plants with Agrobacteria carrying launch vectors. Optimization of Agrobacterium cultivation in AB medium allows direct dilution of the bacterial culture in Milli-Q water, simplifying the infiltration process. Among three tested species of Nicotiana, N. excelsiana (N. benthamiana &#215; N. excelsior) was selected as the most promising host due to the ease of infiltration, high level of reporter protein production, and about two-fold higher biomass production under controlled environmental conditions. Induction of Agrobacterium harboring pBID4-GFP (Tobacco mosaic virus-based) using chemicals such as acetosyringone and monosaccharide had no effect on the protein production level. Infiltrating plant under 50 to 100 mbar for 30 or 60 sec resulted in about 95% infiltration of plant leaf tissues. Infiltration with Agrobacterium laboratory strain GV3101 showed the highest protein production compared to Agrobacteria laboratory strains LBA4404 and C58C1 and wild-type Agrobacteria strains at6, at10, at77 and A4. Co-expression of a viral RNA silencing suppressor, p23 or p19, in N. benthamiana resulted in earlier accumulation and increased production (15-25%) of target protein (influenza virus hemagglutinin).

Optimization and Utilization of Agrobacterium-mediated Transient Protein Production in Nicotiana

Transient protein production in Nicotiana plants based on vacuum infiltration with Agrobacteria carrying launch vectors (Tobacco mosaic virus-based) is a rapid and economic approach to produce vaccine antigens and therapeutic proteins. We simplified the procedure and improved target accumulation by optimizing conditions of bacteria cultivation, selecting host species, and co-introducing RNA silencing suppressors.

最適化と利用の<em&gt;アグロバクテリウム</em中&gt;媒介一過性タンパク質産生<em&gt;タバコ</em

Agrobacterium-mediated transient protein production in plants is a promising approach to produce vaccine antigens and therapeutic proteins within a short ...

AFM-based Mapping of the Elastic Properties of Cell Walls: at Tissue, Cellular, and Subcellular Resolutions

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) micro/nano-indentations, for a JPK&#160;AFM. Specifically, in this protocol we measure the apparent Young&#8217;s modulus of cell walls at subcellular resolutions across regions of up to 100 &#181;m&#160;x 100 &#181;m in floral meristems, hypocotyls, and roots. This requires careful preparation of the sample, the correct selection of micro-indenters and indentation depths. To account for cell wall properties only, measurements are performed in highly concentrated solutions of mannitol in order to plasmolyze the cells and thus remove the contribution of cell turgor pressure.
In contrast to other extant techniques, by using different indenters and indentation depths, this method allows simultaneous multiscale measurements, i.e. at subcellular resolutions and across hundreds of cells comprising a tissue. This means that it is now possible&#160;to spatially-temporally characterize the changes that take place in the mechanical properties of cell walls during development, enabling these changes to be correlated with growth and differentiation. This represents a key step to understand how coordinated microscopic cellular changes bring about macroscopic morphogenetic events.
However, several limitations remain: the method can only be used on fairly small samples (around 100 &#181;m in diameter) and only on external tissues; the method is sensitive to tissue topography; it measures only certain aspects of the tissue&#8217;s complex mechanical properties. The technique is being developed rapidly and it is likely that most of these limitations will be resolved in the near future.

We describe a method to map mechanical properties of plant tissues using an atomic force microscope (AFM). We focus on how to record mechanical changes that take place in cell walls during plant development at wide-field mesoscale, enabling these changes to be correlated with growth and morphogenesis.

細胞壁の弾性特性のAFMベースのマッピング：ティッシュ、携帯電話、および細胞内の解像度で

We describe a recently developed method to measure mechanical properties of the surfaces of plant tissues using atomic force microscopy (AFM) ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

レシオメトリック二分子ビーコンを使用して、生細胞における単エンジニアのRNA転写物のリアルタイムイメージング

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Generation and Purification of Human INO80 Chromatin Remodeling Complexes and Subcomplexes

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 complexes consist of 14 protein subunits including Ino80, a SNF2-like ATPase, which serves both as the catalytic subunit and the scaffold for assembly of the complexes. Functions of the other subunits and the mechanisms by which they contribute to the INO80 complex&#39;s chromatin remodeling activity remain poorly understood, in part due to the challenge of generating INO80 subassemblies in human cells or heterologous expression systems. This JOVE protocol describes a procedure that allows purification of human INO80 chromatin remodeling subcomplexes that are lacking a subunit or a subset of subunits. N-terminally FLAG epitope tagged Ino80 cDNA are stably introduced into human embryonic kidney (HEK) 293 cell lines using Flp-mediated recombination. In the event that a subset of subunits of the INO80 complex is to be deleted, one expresses instead mutant Ino80 proteins that lack the platform needed for assembly of those subunits. In the event an individual subunit is to be depleted, one transfects siRNAs targeting this subunit into an HEK 293 cell line stably expressing FLAG tagged Ino80 ATPase. Nuclear extracts are prepared, and FLAG immunoprecipitation is performed to enrich protein fractions containing Ino80 derivatives. The compositions of purified INO80 subcomplexes can then be analyzed using methods such as immunoblotting, silver staining, and mass spectrometry. The INO80 and INO80 subcomplexes generated according to this protocol can be further analyzed using various biochemical assays, which are described in the accompanying JOVE protocol. The methods described here can be adapted for studies of the structural and functional properties of any mammalian multi-subunit chromatin remodeling and modifying complexes.

This protocol describes a procedure for generating and purifying wild type and mutant versions of the human INO80 chromatin remodeling complex. Epitope tagged versions of INO80 subunits are stably expressed in HEK293 cells, and complete complexes and complexes lacking specific sets of subunits are purified by immunoaffinity chromatography.

発生と人間INO80クロマチンリモデリング複合体とSubcomplexesの精製

INO80 chromatin remodeling complexes regulate nucleosome dynamics and DNA accessibility by catalyzing ATP-dependent nucleosome remodeling. Human INO80 ...