우리는 우리의 방법과 관련된 경험과 관찰의 일부에 기여 귀중한 단백질 샘플을 제공하는 우리의 공동 작업자를 주셔서 감사합니다. 군터 Schmalzing은 친절이 프로젝트에 참여 FR 수있는 기회를 제공했습니다. 바바라 암브러스터, 야곱 브링크 및 Deryck 밀스가 자신의 뛰어난 도움말 및 장비에 대한 입력을위한 감사합니다. 기금은 NIH 부여 HL090630에 의해 제공되었다.

1. 2D 결정화 재판의 그리드 준비 <ol><li> 탄소 코팅 400 메쉬 구리 EM 격자는 부정적인 얼룩으로 준비하고 있습니다. Uranyl 아세테이트는 자주 사용 및 지속적인 격자의 장기 저장을 위해 사용뿐만 아니라 적합성 전에 몇 달 동안 솔루션의 저장 용량 측면에서 얼룩을 제공합니다. 반면, 같은 uranyl의 편대와 같은 다른 부정 얼룩, 우수한 얼룩을 제공하면서는, 갓 1 만들었해야합니다. 2D 결정화 실험의 심사에 사용되는 격자의 다수의 빠른 준비, 부정적인 얼룩의 수정된 버전이 사용됩니다. 샘플 2 μL의 볼륨은 탄소 대상 EM 그리드에 pipetted 60 S.위한 incubated입니다 이것은 왓먼의 찢어진 조각 # 4 필터 용지 (그림 1, 비디오)와 가장자리에서 모래 바닥을 따라, 그리고 1 % uranyl 아세테이트 2 μL를 즉시 적용되고있는 다시 30 후 격자의 가장자리에서 blotted 아르 S. 필터 종이의 찢어진 가장자리와 가장자리에있는 격자를 감동하면 proteoliposomes 제거하지 않고 액체의 최적의 제거를 보장합니다. 또한, 그리드의 가장자리에 건조가 탄소 필름의 향상된 보존을 보장합니다. 섬세한 탄소 필름의 파손은 샘플 접착을 방지하고 샘플의 부정 확한 표현이 발생할 수 있으므로주의가, 준비와 격자의 취급에 촬영해야합니다. 5 μL의 전통적으로 큰 샘플 볼륨이되었고 정기적으로 그리드 준비에 사용되는 반면, 소중한 샘플 2 μL 또는 2 이하로 볼륨을 감소시켜 저장할 수 있습니다. </li><li> 가장 큰 가능한 세포막을 생산하기 위해, 우리의 샘플 중 일부는 글리세롤이나 자당 높은 농도 (10~20%)는 투석 버퍼에 존재해야합니다. 글리세롤이나 자당이 높은 점성이고 제대로 버퍼를 관통하고, 따라서 불완전 세포막을 얼룩으로부터 uranyl 아세트산을 방지로 이것은 그리드의 준비에 부정적인 영향을 미칠 수 있습니다. 따라서 가능한 격자가 드러나지 않습니다. 어느 버퍼가 글리세롤 / 자당 무료 버퍼 (표시되지 않음), 또는 격자의 샘플 및 교체의 원심 분리에 의해 교환 수가와 유사한 부정적인 얼룩 전에 몇주기 버퍼 또는 하나의 글리세롤 / 자당 무료 버퍼로 씻어 수 있습니다 cryo - EM 3,4에 사용되는 기술. </li></ol> 2. EM으로 2D 결정화 평가판을 평가 <ol><li> 샘플 홀더의 유형에 따라 하나 또는 여러 개의 격자 중 하나가로드됩니다. 중간 배율로 여기라고한다 2 - 10K의 확대는 실험실 노트북 5주의 촬영입니다 세포막, 형태, 및 크기의 분산의 평균 유통 및 범위의 첫 인상에 대한 수 있습니다. CCD 카메라는 필름에 사용할 수없는 경우 적합 영역, CCD 카메라의 도움으로 대표 개요로 기록 또는 있습니다. </li><li> 샘플 / 그리드 전체의 개요가 원하는 경우 약 400 800x의 범위에서 낮은 배율은 시간에 사용됩니다. 모든 그리드와 고용 없지만, 낮은 배율은 여러 측면 측면에서 그리드 준비의 평가에 도움이 귀중한 정보를 제공합니다 : 부정 얼룩 및 탄소 필름, 그리드에서 샘플 농도, 그리고 아마도 고르지 proteoliposome 배포판의 가능성이 일부 파손 모두. 개인 그리드 사각형은 쌍안경이나 CCD 카메라로 볼 수 있습니다. 일부 EMS과 함께 더 높은 배율에서 나중에 검사에 대한 리콜 수있는 특정 관심의 위치를​​ 저장 할 수 있습니다. </li><li> 관심 그리드 영역 중 낮은 또는 중간 배율에서 확인되었습니다되면, 배율은 약 50K - 60K로 변경됩니다. 알려진 경우 30K로 낮은 80K처럼 높은 배율은, 멤브레인, 크리스탈과 단위 셀 크기에 따라 것은 사용됩니다. 30 - 80K의 배율 범위는 2D 결정에 대한 심사의 목적은 높은 배율로 불리는 것입니다. 포커싱 이미지 / 사진 설정하거나, 관심있는 지역의 주변에 이후의 스위치와 함께, 낮은 복용량 설정의 초점 설정에서 하나 발생합니다. <ol class="lalpha"><li> 관심 분야가 실제로 막 여부에 모호함의 경우, 샘플 proteoliposomes, 운모, 또는 기타 유물의 크기 탄소 필름 검사입니다. 이러한 목적으로, 가장자리는 전형적인 접는 조직 형태를 공개. </li><li> 이제 관심의 영역은 CCD 카메라와 함께 검사합니다. CCD 이미지는 막의 크기, 단백질 또는 단위 셀 크기에 따라 30K - 80K 확대 수집하거나, 결정 영역을 알려져 있습니다. 작은 및 / 또는 대부분 소수성 막 단백질의 격자는 CCD 이미지 자체의 시각적 평가에 의해 반드시 표시되지 않습니다. 어느 전체 이미지가 온라인 푸리에 변환 (FT, 또는 빠른 FT - FFT)에 사용됩니다. 주문 배열 작은 경우이 FT는 그러나, 소음들을 상당히 포함됩니다. 따라서, 감소 박스 이미지 크기는 smal의 향상된 신호 대 잡음 비율에 대한 수ler 크리스탈 쉽게 식별. 이러한 목적으로, 상자는 이미지를 통해 이동하고 라이브 FT는 평가입니다. 빔의 강도 / 밝기는 낮은 복용량 샘플 조건뿐만 아니라, 마음에 CCD 카메라 설정을 유지 조정됩니다. 사용 CCD 카메라에 따라 살아있는 FT의 감마는 주문한 배열의 최적의 식별을 위해 조정됩니다. 지나치게 높은 값을 소음 공헌으로 인해 장소를 가리 수 있고, 지나치게 낮은 감마 값을 식별되는 약한 장소를 방지할 수 있습니다. 이러한 약한 명소 거의 소음 수준 위의 FT의 명소로 작은 결정 배열에 의한 것입니다. 높은 해상도에서 데이터가 샘플 6 소수의 수집되어 있지만, 부정적인 스테인드 차원 결정의 일반적으로 해상도가 제한되거나 약 15A 해상도보다 나은 것으로 예상해서는 안됩니다. 약 -400 NM의 defocus 아니라 함께 명소 이상의 1-3 명령은 쉽게 식별 것으로 예상된다. 샘플은 일반적으로 cryo - EM 데이터 수집이 최고 달성 해상도의 적절한 표시를 줄 것이다로서, 해상도에 대해 평가하지 않습니다. 장소 가능한 mosaicity의 선명도는하지만 설명되어 있습니다. </li></ol></li><li> 다른 세포막뿐만 아니라 멤브레인 morphologies은 높은 배율에서 주문 평가하고 있습니다. 작은보다 큰 proteoliposomes 또는 막 패치가 가장 유망한 분야를 포함할 수 있듯이, 시작 또는 2D 결정화 실험의 중간 단계에 특히 중요합니다. 2D 결정의 매우 낮은 비율은 주문한 배열의 초기 식별이 자주 크기와 품질 2,4,7의 급속한 발전으로 이어질 것입니다 때문에 이미지의 다수의 이미지 수집 및 FT, 또는 광학 회절을 필요합니다. </li></ol> 3. 대표 결과 이상적으로 주문 proteoliposomes가 쉽게 알아볼, 날카로운 명소를 표시합니다. 대형 잘 명령 결정을 쉽게 CCD 이미지 또는 micrographs의 광학 회절의 온라인 FT에 의해 식별됩니다. 예를 들어 크기가 몇 미크론까지 아르 18kDa의 작은 막 단백질의 2D 수정을 보여줍니다. FT에 얼룩이 쉽게 식별하고 날카로운 있습니다. 라이브 - FT 상자의 움직임은 격자가 mosaicity없이 지속 것을 보여줍니다. 보다 광범위한 가용성 도메인과 큰 단백질의 격자는 EM의 작은 화면에서 확인할 수 있습니다. CCD 이미지 수집 및 FT 더 나은 평가 수단을 제공하고, 예를 들어, 가능한 mosaicity (쇼 FT)에 대한 정보를 얻을 필요가 있습니다. 주문되지 않은 proteoliposome의 FT을 계산할 때, 소음이 처음 장소에 대한 오해하실 수 있습니다. 라이브 FT에 대한 상자가 이동하는 동안 그러나, 반점이 사라집니다. 반면에, 작은 배열, 의심 결정화와 라이브 FT는 이미지 영역도 약간 이동하면 고정 남아있는 그들의 반점을 가지고 있습니다. 또한, 이러한 작은 결정은 일반적으로 동일한 단위 셀 크기를 가지고 인식 될 수 있으며, 다른 FTs의 명소 간의 거리는 특정 크기의 원형과 같은 다양한 방법으로 측정할 수 있습니다. 지질 결정은 별개의 격자 형태와 FT를 표시합니다. 그것은 초기 실험에서 강수량이 발생하는 일반적입니다. reconstitution없이 여기 단백질 강수량하지만 작은 지질 집계에서 구별해야합니다. 낮은 배율에서 precipitates 것으로 나타날 샘플 자주 높은 배율에서 볼 때 지질 집계로 판명. 30 - 50K에서 검사시 이러한 어두운 구조의 가장자리 그들은 단백질없이 강수량과 세포막의 구성을 밝혀. 지질 집계는 다음 실험에 큰 세포막 크기의 증가 수도 있습니다 이러한 중요한 관찰합니다. 평가 샘플에서 가난한 결과는 때로는 적절한 빠른 검사을 방지 낮은 멤브레인 농도에 연결되어 있습니다. 이것은 자주 투석에 의해 2D 결정에 대한 높은 단백질 농도를 사용하여 극복할 수 있습니다. 또는 안정이 저장되는 동안 Eppendorf 튜브의 하단에 며칠 동안 정착에 남아있을 수 있습니다. 어떤 경우에는 세포막의 신속, 또는 거의 인스턴트 정착이 발생하여 튜브 바닥에서 pipetting하면 그리드에서 높은 막 밀도가 높아집니다. 또 다른 훨씬 빠른 방법은 튜브의 바닥에서 이후의 샘플링과 샘플 원심 분리 (1~3분에 대한 3000-8000 rpm으로)입니다. 최적의 조건 하에서 샘플은 2D 결정의 큰 비율을 포함합니다. 그것은 가장 크고 가장 잘 주문한 2D 결정이 데이터에 대한 시각이 선택되어 모음으로, 점막의 균일한 모양 목표 필요가 없습니다. 결정화 실험이뿐만 아니라 샘플을 사용하는 경우 반복하는 경우 샘플을 이런 종류의 쉽게 인식됩니다고해상도 이미지의 최대 개수에 결과 cryo - EM 데이터 수집. <img src="/files/ftp_upload/1846/1846fig1.jpg" alt="그림 1" /> 그림 1.이 그림은 왓먼의 찢어진 조각 # 4 필터 종이 그리드의 가장자리를 모래 바닥 보여줍니다.

<ol>
	<li>Johansen, B. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+field+electron+microscopy+of+biological+specimens+V.+A+low+dose+pre-irradiation+procedure+reducing+beam+damage.">Bright field electron microscopy of biological specimens V. A low dose pre-irradiation procedure reducing beam damage.</a> Micron. 7, 145-156 (1976).</li><li>Schmidt-Krey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+crystallography+of+membrane+proteins:+Two-dimensional+crystallization+and+screening+by+electron+microscopy.">Electron crystallography of membrane proteins: Two-dimensional crystallization and screening by electron microscopy.</a> Methods. 41, 417-426 (2007).</li><li>Wang, D. N., K&#252;hlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+electron+crystallography+of+light-harvesting+chlorophyll+a/b-protein+complex+in+three+different+media.">High-resolution electron crystallography of light-harvesting chlorophyll a/b-protein complex in three different media.</a> J Mol Biol. 217, 691-699 (1991).</li><li>Schmidt-Krey, I., Rubinstein, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electron+cryomicroscopy+of+membrane+proteins:+specimen+preparation+for+two-dimensional+crystals+and+single+particles.">Electron cryomicroscopy of membrane proteins: specimen preparation for two-dimensional crystals and single particles.</a> Micron. , (2010).</li><li>Schmidt-Krey, I., Mutucumarana, V., Haase, W., Stafford, D. W., K&#252;hlbrandt, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+of+human+vitamin+K-dependent+&amp;gamma;-glutamyl+carboxylase.">Two-dimensional crystallization of human vitamin K-dependent &amp;gamma;-glutamyl carboxylase.</a> J Struct Biol. 157, 437-442 (2007).</li><li>Trachtenberg, S., DeRosier, D. J., Zemlin, F., Beckmann, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-helical+perturbations+of+the+flagellar+filament:+Salmonella+typhimurium+SJW117+at+9.6+&amp;Aring;+resolution.">Non-helical perturbations of the flagellar filament: Salmonella typhimurium SJW117 at 9.6 &amp;Aring; resolution.</a> J Mol Biol. 276, 759-773 (1998).</li><li>Zhao, G., Johnson, M. C., Schnell, J. R., Kanaoka, Y., Irikura, D., Lam, B. K., Austen, K. F., Schmidt-Krey, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-dimensional+crystallization+conditions+of+human+leukotriene+C4+synthase+requiring+a+particularly+large+combination+of+specific+parameters.">Two-dimensional crystallization conditions of human leukotriene C4 synthase requiring a particularly large combination of specific parameters.</a> J Struct Biol. 169, 450-454 (2010).</li><li>Cheng, A., Leung, A., Fellmann, D., Quispe, J., Suloway, C., Pulokas, J., Abeyrathne, P. D., Lam, J. S., Carragher, B., Potter, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+automated+screening+of+two-dimensional+crystals.">Towards automated screening of two-dimensional crystals.</a> J Struct Biol. 160, 324-331 (2007).</li><li>Vink, M., Derr, K. D., Love, J., Stokes, D. L., Ubarretxena-Belandia, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+strategy+to+screen+2D+crystallization+trials+of+membrane+proteins.">A high-throughput strategy to screen 2D crystallization trials of membrane proteins.</a> J Struct Biol. 160, 295-304 (2007).</li></ol>

샘플 적절한 평가는 세포막의 충분한 숫자의 신중 평가가 필요합니다. 예를 들어, 180여 군데 proteoliposomes 밖으로 낮은 % 2로 결정 배열 샘플 2D 결정화 조건 7 빠르고 최적화에 대한 중요한 정보를했다. 단백질의 일부​​ 가끔 침전이 발생하지만, 단백질의 침전이 발생하면, 그리드는 낮은 배율에서 검사 후 추가 검사에서 버려진 수 있습니다. 100 개 미만 NM의 비록 아?...

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Comment</th>
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<td>400-mesh copper TEM grids coated with carbon film</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>forceps: regular and anti-capillary </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Dumont #5 and Dumont N5AC or similar</td>
</tr>
<tr>
<td>Micropipette and pipette tips</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Whatman #4 filter paper</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1% uranyl acetate</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Dialysis sample to be screened for 2D crystals</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
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<td>Glycerol/sucrose-free dialysis buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Optional</td>
</tr>
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<td>JEOL-1400 transmission electron microscope (TEM)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>similar 80 &#x2013; 120kV TEM equipped with an Lab6 or tungsten filament and film and/or CCD cameras (Gatan Orius SC1000 and/or UltraScan1000 CCD cameras and Gatan Digitial Micrograph software package or Tietz cameras (TVIPS)) </td>
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assessing two-dimensional crystallization trials of small membrane proteins for structural biology studies by electron crystallography

전자 crystallography 중 하나 또는 또는 구조 기능 막 단백질의 질문에,뿐만 아니라 수용성 단백질을 연구 세 가지 차원 결정화 및 X - 선 crystallography와 함께 사용할 수있는 방법으로 진화했다. 전송 전자 현미경에 의해 2 차원 (2D) 결정에 대한 심사는 (EM) 최적화, 그리고 cryo - EM하여 고해상도 데이터 수집에 대한 샘플을 선택 찾는 중요한 단계입니다. 여기 우리는 잠재적으로 결정화 조건의 최적화에 대한 중요한 정보를 제공 수, 대형 및 주문뿐만 아니라, 작은 2D 배열을 모두 식별에 근본적인 단계를 설명합니다. EM에서 서로 다른 배율과 협력함으로써, 중요한 매개 변수의 범위에있는 데이터가 얻어진다. 낮은 배율은 형태학 및 멤브레인 크기에 귀중한 자료를 제공합니다. 높은 배율에서 가능한 순서 및 2D 크리스탈 크기가 결정됩니다. 이러한 맥락에서, 그것은 CCD 카메라와 온라인 푸리에 변환이 순서와 크기 proteoliposomes을 평가하기 위해 더 높은 배율에서 사용하는 방법에 설명되어 있습니다. 막 단백질의 2D 결정이 가장 일반적으로 투석에 의해 reconstitution에 의해 성장하는 동안, 심사 기술 monolayers, 기본 차원 결정의 도움으로 제작 결정에 동일하게 적용되며, 수용성 단백질의 배열을 명령했다. 또한, 여기에서 설명한 방법 작은 단백질이 큰 단백질 우리의​​ 예제와 격자로 식별에있는 치료의 동일한 금액을 요구에도 소규모의 2D 결정뿐만 아니라 큰 멤브레인 단백질에 대한 심사에 적용되는 더 쉽게 식별할 수있을 심사의 이전 단계에서.

Watch this Scientific Journal Video about Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography at JoVE.com

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

주문한 막 단백질 배열의 형성을위한 2 차원 (2D) 결정화 실험을 평가하는 것은 전자 crystallography에 매우 중요하고 어려운 작업입니다. 90kDa - 여기에 대한 심사와 15의 범위에서 주로 작은 막 단백질의 2D 결정을 식별하는 우리의 접근 방식을 설명합니다.

전자 Crystallography에 의해 구조 생물학 연구에 대한 작은 막 단백질의 2 차원 결정화 평가판을 평가

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

assessing-two-dimensional-crystallization-trials-small-membrane

Research

JoVE Journal

Biology

11.2K 조회수.  Georgia Institute of Technology. 주문한 막 단백질 배열의 형성을위한 2 차원 (2D) 결정화 실험을 평가하는 것은 전자 crystallography에 매우 중요하고 어려운 작업입니다. 90kDa - 여기에 대한 심사와 15의 범위에서 주로 작은 막 단백질의 2D 결정을 식별하는 우리의 접근 방식을 설명합니다.

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

Microcontact Printing of Proteins for Cell Biology

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. Development of microcontact printing by the Whitesides group (<a href="http://gmwgroup.harvard.edu/">http://gmwgroup.harvard.edu/</a>) in the mid-1990s revolutionalized this field by making microelectronics/microfabrication techniques accessible to laboratories focused on the life sciences. Initial implementations of this method used polydimethylsiloxane (PDMS) stamps to create patterns of functionalized chemicals on material surfaces1. Since then, a range of innovative approaches have been developed to pattern other molecules, including proteins2. This video demonstrates the basic process of creating PDMS stamps and uses them to pattern proteins, as these steps are difficult to accurately express in words. We focus on patterning the extracellular matrix protein fibronectin onto glass coverslips as a specific example of patterning.
An important component of the microcontact printing process is a topological master, from which the stamps are cast; the raised and lowered regions of the master are mirrored into the stamp and define the final pattern. Typically, a master consists of a silicon wafer coated with photoresist and then patterned by photolithography, as is done here. Creation of masters containing a specific pattern requires specialized equipment, and is best approached in consultation with a fabrication center or facility. However, almost any substrate with topology can be used as a master, such as plastic diffraction gratings (see Reagents for one example), and such serendipitous masters provide readily available, simple patterns. This protocol begins at the point of having a master in hand.

Microcontact printing is used extensively to pattern proteins and other molecules on material surfaces. We demonstrate the basic steps of this process, stamping patterns of fibronectin onto glass.

세포 생물학을위한 단백질의 Microcontact 인쇄

The ability to pattern proteins and other biomolecules onto substrates is important for capturing the spatial complexity of the extracellular environment. ...

연구

생물학

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

Lipidic Mesophases를 사용하여 구조 결정을위한 막 단백질 Crystallizing

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

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 - 선 Crystallography에 대한 단백질 결정화

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.

Lipidic Mesophases의 막 단백질의 결정화

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.

Lipidic Bicelle 방법을 사용하여 막 단백질의 높은 처리량 결정

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.

Lipidic Mesophases의 막 단백질의 높은 처리량을 결정하기위한 로봇의 사용

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

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

Macromolecular의 결정학에 의해 구조 결정에 대한 Lipidic Mesophases에서 재배 막 단백질의 수확 및 Cryo 냉각 크리스털

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Visualization of ATP Synthase Dimers in Mitochondria by Electron Cryo-tomography

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as cells, organelles, membrane vesicles, or viruses at molecular detail. To achieve this, the aqueous sample is rapidly vitrified in liquid ethane, which preserves it in a close-to-native, frozen-hydrated state. In the electron microscope, tilt series are recorded at liquid nitrogen temperature, from which 3D tomograms are reconstructed. The signal-to-noise ratio of the tomographic volume is inherently low. Recognizable, recurring features are enhanced by subtomogram averaging, by which individual subvolumes are cut out, aligned and averaged to reduce noise. In this way, 3D maps with a resolution of 2 nm or better can be obtained. A fit of available high-resolution structures to the 3D volume then produces atomic models of protein complexes in their native environment. Here we show how we use electron cryo-tomography to study the in situ organization of large membrane protein complexes in mitochondria. We find that ATP synthases are organized in rows of dimers along highly curved apices of the inner membrane cristae, whereas complex I is randomly distributed in the membrane regions on either side of the rows. By subtomogram averaging we obtained a structure of the mitochondrial ATP synthase dimer within the cristae membrane.

We present a protocol of how to collect and process electron cryo-tomograms of whole mitochondria. The technique provides detailed insights into the structure, function, and organization of large membrane protein complexes in native biological membranes.

전자 곳을 알아내는-단층 촬영에 의한 미토콘드리아에서 ATP 합성 효소 이량 체의 시각화

Electron cryo-tomography is a powerful tool in structural biology, capable of visualizing the three-dimensional structure of biological samples, such as ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

고립 된 미토콘드리아의 유동 세포 계측법에 의해 외부 막 단백질의 면역

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Methods Article

전자 Crystallography에 의해 구조 생물학 연구에 대한 작은 막 단백질의 2 차원 결정화 평가판을 평가