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<p>Hi Aninda,</p>
<p>regarding to bacterial membrane enzymes that potentially form dimers, with each dimer having a solved molecular weight under 100 kDa. Despite the inherent challenges posed by their small size, similar cases have been successfully addressed in cryo-EM studies.</p>
<p>Small membrane proteins (MPs) under 100 kDa present challenges due to their low natural abundance, high hydrophobicity, and instability in conformation and composition. Additionally, factors such as low contrast and a poor signal-to-noise ratio (SNR) critically impact the quality and resolution of analyses.</p>
<p>In cryo-EM, differentiating protein regions from the background—such as detergents or lipid nanodiscs in amorphous ice—is crucial. This differentiation is impeded by the significant solvent volume fraction, complicating image alignment and 3D reconstruction.</p>
<p>The low molecular mass of small proteins results in fewer electrons scattered, leading to a weaker signal. The SNR further degrades when proteins are embedded in thicker ice regions, introducing additional background noise.</p>
<p>Effective cryo-EM analysis necessitates that proteins be homogeneous and stable in their conformation to ensure that collected images represent consistent conformational states, allowing for accurate alignment and averaging. The variability in particle orientation and conformation among small proteins often leads to poor alignment, compromising the quality of the averaged image and the resolution of the final 3D density map.</p>
<p>Strategic Innovations and Solutions in general:</p>
<ul>
<li>Optimization of Vitrification Conditions: refining the vitrification processes to control ice thickness and enhance contrast, and employing advanced detectors (e.g., K3, Falcon) for optimal results with phase plate and energy filter.</li>
<li>Innovations in Detergents and Nanodiscs: exploring new formulations and structures to stabilize these proteins within their native conformations.</li>
<li>Advanced Software and Image Processing Algorithms: utilizing sophisticated algorithms, including blush regularization in RELION 5 and non-uniform refinement in CryoSPARC, to enhance contrast and alignment for accurate 3D reconstructions.</li>
<li>Use of Rigid Markers and Grid Improvements: investigating the use of rigid markers out of membrane can improve the image alignment quality of side views lead to an better 3D reconstruction map.</li>
<li>Employing grids coated with gold and graphene to reduce beam-induced motion and interaction issues to have better particle distribution in the thin ice area.</li>
</ul>
<p>For further insights,there is also a review article published last year that discusses approaches to solving sub-100 kDa membrane proteins using cryo-EM: <a href="https://urldefense.com/v3/__https://doi.org/10.1016/j.jsb.2023.107959__;!!Mih3wA!BqAyE3_xYLoBT2inNZc4hDweDyBUhT_uN9EwV_Hd1gS2KcsX5_wCnt8ONc9CETdCHKlJs6xMFZ9xHvMLrr2bfH5kutsLBnz1YTY$">https://doi.org/10.1016/j.jsb.2023.107959</a>.</p>
<p>Best regards,</p>
<p>Lifei</p>
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<div id="GroupWiseSection_1712593565304_Lifei.Fu@biologie.uni-regensburg.de_D08D7A5015800000AF446463B305BE80_" class="GroupWiseMessageBody"><span class="GroupwiseReplyHeader"><br>>>> Guillaume Gaullier <guillaume.gaullier@kemi.uu.se> 08.04.2024, 12:47 >>><br></span> Hi Aninda,
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<div class="">Here is a recent review on cryoEM of small proteins: <a class="" href="https://urldefense.com/v3/__https://doi.org/10.1016/j.str.2024.03.003__;!!Mih3wA!HjneFU3_I0LNA5Ecsh4kLh0y-aemd0SeU9_j156zgWRzsjVBc3UjwUKC4a_IbFBNlxbLfRt0INvqDSUWJasoXaTuv7SrZlB_$">https://doi.org/10.1016/j.str.2024.03.003</a></div>
<div class="">It is not specific to membrane proteins, but might answer some of your questions or give you pointers to look further.</div>
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<div class="">This other article with a systematic exploration of data collection parameters for GPCRs might be more directly relevant to you, even though GPCRs are larger than the proteins you describe: <a class="" href="https://urldefense.com/v3/__https://doi.org/10.1038/s41467-021-24650-3__;!!Mih3wA!HjneFU3_I0LNA5Ecsh4kLh0y-aemd0SeU9_j156zgWRzsjVBc3UjwUKC4a_IbFBNlxbLfRt0INvqDSUWJasoXaTuv0toUZGm$">https://doi.org/10.1038/s41467-021-24650-3</a>
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<div class="" style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px; word-wrap: break-word; -webkit-nbsp-mode: space; line-break: after-white-space;">
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<div style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px;"> </div>
<div style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px;">When you don’t get answers on this 3DEM list, I suggest to also search the archives of the CCPEM list: <a class="" href="https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/webadmin?A0=CCPEM__;!!Mih3wA!HjneFU3_I0LNA5Ecsh4kLh0y-aemd0SeU9_j156zgWRzsjVBc3UjwUKC4a_IbFBNlxbLfRt0INvqDSUWJasoXaTuv7veKZys$">https://www.jiscmail.ac.uk/cgi-bin/webadmin?A0=CCPEM</a></div>
<div style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px;">And if you still don’t find anything relevant, of course you are welcome to post your questions to the CCPEM list as well. Some people follow both lists (I do), but I don’t know how much the two populations overlap, so for certain questions you might reach more people with potential answers on the other list.</div>
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<div style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px;">I hope this helps,</div>
<div style="color: rgb(0, 0, 0); font-family: Helvetica; font-size: 12px; font-style: normal; font-variant-caps: normal; font-weight: normal; letter-spacing: normal; text-align: start; text-indent: 0px; text-transform: none; white-space: normal; word-spacing: 0px; -webkit-text-stroke-width: 0px;"><br class="Apple-interchange-newline">Guillaume</div>
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<div class="">On 6 Apr 2024, at 22:46, Aninda Dutta <<a class="" href="mailto:anindadutta86@gmail.com">anindadutta86@gmail.com</a>> wrote:</div>
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<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">Dear 3DEM list members,</span></div>
<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">I have two small bacterial membrane enzymes (sub-50 kDa) that I want to work with. Both of them are known to dimerize under certain conditions and I want to understand the conformational dynamics of these systems under native lipid environments (Liposome, nanodiscs) using CryoEM.</span></div>
<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">Regarding that, I have some questions to ask all of you - </span></div>
<ol class="" style="color: rgb(36,36,36); font-size: 15px; margin-top: 0px; margin-bottom: 0px;" start="1">
<li class="" style="font-size: 11pt;">
<div class="" style="border: 0px; font-style: inherit; font-variant: inherit; font-weight: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">How are we as a community doing with sub-50 kDa CryoEM without using Fabs or other nanobodies?</span></div>
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<div class="gmail-x_elementToProof" style="border: 0px; font-style: inherit; font-variant: inherit; font-weight: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">What are the challenges, and can we come over it?</span></div>
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<div class="gmail-x_elementToProof" style="border: 0px; font-style: inherit; font-variant: inherit; font-weight: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">Tell me any suggestions or tips you may have before starting this project.</span></div>
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<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;"> </span></div>
<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">Last but not the least, I am open to any interesting collaborations.</span></div>
<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;"> </span></div>
<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">Best,</span></div>
<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">Aninda</span></div>
<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;"> </span></div>
<div class="gmail-x_elementToProof" style="border: 0px; font-variant-numeric: inherit; font-variant-east-asian: inherit; font-variant-alternates: inherit; font-stretch: inherit; font-size: 11pt; line-height: inherit; font-kerning: inherit; font-feature-settings: inherit; margin: 0px; padding: 0px; vertical-align: baseline;"><span class="" style="font-family: arial, sans-serif;">---------------------</span></div>
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<div class=""><strong class="" style="color: rgb(230,145,56);"><span class="" style="font-family: garamond,times new roman,serif; font-size: large;"><em class="">Aninda Dutta</em></span></strong></div>
<div class=""><span class="" style="font-family: 'comic sans ms', sans-serif; font-size: small;"><em class=""><strong class="">UVA Chem</strong> Graduate Student </em></span><span class="" style="font-family: 'comic sans ms', sans-serif; font-size: small;"><em class=""><br class=""></em></span></div>
<div class=""><span class="" style="font-family: 'comic sans ms', sans-serif; font-size: small;">University of Virginia</span></div>
<div class=""><span class="" style="font-family: 'comic sans ms', sans-serif; font-size: small;">Charlottesville, VA</span></div>
<div class=""><span class="" style="font-family: 'comic sans ms', sans-serif; font-size: small;"> </span></div>
<div class=""><span class="" style="font-family: comic sans ms,sans-serif; color: rgb(153,0,255); font-size: small;"><img class="" src="https://ci3.googleusercontent.com/mail-sig/AIorK4yMZgG-3qQiCpz2B8vheUr9_TfYSWxaLOxB_YAb6rEC8_hjaG5K3Ax2KPgfzw09JSBpoMOa7Kiof7gp" width="200" height="58"><br class=""></span></div>
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