And seriously, Google is basically my #1 resource 🙂 So a big part of grad school is Googling! (and critically evaluating what you find!). A lot of the time Google will take me to some of these sites – other times I go straight to these sites, often through bookmarked tabs. But no matter how I find them, I’m grateful for them!
Oh – and if you have a super specific question you can’t find the answer to anywhere, try asking on Twitter or Reddit. It’s worked great for me many a time!
Note: a lot of these resources have a lot more features than I’ve used (or than I even know about!) so I’m going to highlight just what I’ve used them for – but of course feel free to explore and use even more! Note that the order within sections is mostly un-intentional (not ranking them or anything!)
Resources for learning
First a couple pages on my site that might help – Brisources you might say…:
- “experimental techniques” page where you can find links to posts on things like SDS-PAGE, PCR, western blots, etc. https://thebumblingbiochemist.com/lets-talk-science/techniques/
- a glossary of de-jargonnized biochemistry jargon https://thebumblingbiochemist.com/glossary-2/
- protein purification page: https://thebumblingbiochemist.com/lets-talk-science/protein-purification/
- one on mathy stuff (dimensional analysis, logarithms, standard deviation, etc.). https://thebumblingbiochemist.com/lets-talk-science/math-y-stuff/
- one on metabolism (making and breaking molecules): https://thebumblingbiochemist.com/lets-talk-science/metabolism-making-and-breaking-molecules/
- amino acids (protein letters) https://thebumblingbiochemist.com/lets-talk-science/amino-acids/
- grad-school related posts: https://bit.ly/gradschoolposts
my YouTube channel playlists: https://www.youtube.com/channel/UChzTIByeodToYsKiNiwKQrw/playlists
Now that I’ve gotten the kinda awkward self-plugging out of the way, here are what other people have to say! Starting with more general resources and then more specific things, like tools for working with proteins, DNA and RNA, and some structural biology stuff
Lehninger Principles of Biochemistry by David L. Nelson and Michael M. Cox. MY ALL TIME FAVORITE
another great biochemistry textbook is Biochemistry: An Integrative Approach by John Tansey. I especially like the cool connections to real-world things which makes for a very relatable read.
for organic chemistry, I like Organic Chemistry Made Ridiculously Simple by Gene A. Davis
Cell Biology by the Numbers by Rob Phillips and Ron Milo. I was introduced to this book when I got to take a course by Dr. Philipps and it was awesome! I really like this book because it gives you a sense of sizes and dimensions and speeds of various cellular processes so you can really put abstract concepts in context. It emphasizes back-of-the-envelope calculations and makes things simple. There’s a print edition but you can also read it for free online http://book.bionumbers.org/
Organic Chemistry With a Biological Emphasis by Tim Soderberg. I haven’t had a chance to check this out much but it’s a free o-chem text book with a biochem angle. Totally free – including practice problems and solutions. https://sites.google.com/morris.umn.edu/timsoderberg/home/organic-chemistry-with-a-biological-emphasis
Bionumbers: The Database of Useful Biological Numbers: this site is a go-along to the Cell Biology by the Numbers book. It has tons and tons of statistics for things you never knew you wanted to know (like length of an eye blink) as well as core key values like cell diameters, replication times, etc. https://bionumbers.hms.harvard.edu/search.aspx
They have a cheat sheet with key numbers: https://bionumbers.hms.harvard.edu/keynumbers.aspx
LabXchange: this site run by Harvard and amgen hosts a variety of free educational content (and even shares some of my stuff). They have a range of content – videos, text, infographics, and, coolest of all, interactive virtual labs. Teachers can use it to build course content and pathways but students can just use it freely as well and there are a range of pre-made “clusters” which are kinda like mini-courses that content content from multiple different collaborators. https://www.labxchange.org/explore
The Medical Biochemistry Page: this website has come in really handy for a lot of my posts where there’s a medical connection as well as a lot of my amino acid posts. They do a great job going in-depth on biochemical pathways and key players as well as how mutations in those players can cause problems https://themedicalbiochemistrypage.org/
Chemguide: I LOVE this site for whenever I’m confused about anything chemistry-y! https://www.chemguide.co.uk/
Chemistry LibreTexts: they have a bunch of really great free open-access text content. https://chem.libretexts.org/
HyperPhysics: hosted by GeorgiaState University’s Department of Physics and Astronomy. I tend to end up here a lot via Google but here’s their main link http://hyperphysics.phy-astr.gsu.edu/hbase/index.html
Addgene blog, especially Plasmids 101: everything you ever wanted to know about plasmids and molecular cloning including details and comparisons of various techniques. https://info.addgene.org/plasmids-101-topic-page
IUBMB-Nicholson Metabolic Maps: you can download and print or they have a really cool interactive version where you can explore and get information about the various components https://iubmb.org/resources/iubmb-nicholson-metabolic-maps/
International Union of Biochemistry and Molecular Biology (IUBMB) website: lots of great resources, information about meetings, as well as fellowship information for biochemists around the world https://iubmb.org/
EMBL-EBI Mechanism and Catalytic Site Atlas So cool! It shows you the stepwise mechanism and even the active site involvement. https://www.ebi.ac.uk/thornton-srv/m-csa/
ThermoFisher Protein Expression Handbook: discusses various strategies for expressing proteins and is a good place to start for people wanting to get into recombinant expression https://www.thermofisher.com/us/en/home/life-science/protein-biology/protein-biology-learning-center/protein-biology-resource-library/protein-expression-handbook.html
Guide to Baculovirus Expression Vector Systems (BEVS) and Insect Cell Culture Techniques https://tools.thermofisher.com/content/sfs/manuals/bevtest.pdf
Cytiva (previously GE) Principles & Methodology Handbooks (all free to download without registering or anything although they do include product information) – https://www.cytivalifesciences.com/en/us/support/handbooks
There are lots of good ones but the Strategies for protein purification handbook is a great place to start for people interested in purification https://cdn.cytivalifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=15680
They also have good information about troubleshooting!
Khan academy: They have a lot of great videos on core concepts and I especially like when they work out examples. They have videos on tons of different subjects and at various skill levels. https://www.khanacademy.org/
Bozeman Science has a lot of great videos on YouTube about various science topics: https://www.youtube.com/user/bozemanbiology
Tyler DeWitt has really great ones on chemistry with worked-out examples and stuff: https://www.youtube.com/user/tdewitt451
I’ve been watching a couple that I really like:
Chem 51A. Organic Chemistry taught by Professor Susan King
Chem 128: Introduction to Chemical Biology taught by Professor Gregory Alan Weiss
Amanda L. Jonsson, Mark A.J. Roberts, J.L. Kiappes, Kathryn A. Scott; Essential chemistry for biochemists. Essays Biochem 31 October 2017; 61 (4): 401–427. doi: https://doi.org/10.1042/EBC20160094
this is a must-read for biochemists!
Resources for lab-bing – some practical tools for use when designing & setting up experiments
For a lot of these, it’s good to know how to do it without one of these calculators, but they can be big time-savers AS LONG AS YOU KNOW HOW TO DO IT! Just like how you should know how to add 2 and 2 before using a standard calculator! They can also help prevent stupid math mistakes that can be costly in the lab!
OpenWetWare Protocols: this is a wiki with tons of protocols including recipes for making reagents that typically come in kits so you can DIY it. The site has other features as well but I’ve only used it for the protocols. https://openwetware.org/wiki/Protocols
Cold Spring Harbor Protocols: this has tons of protocols. Some are free for everyone, others require that you or your institution are subscribed http://cshprotocols.cshlp.org/
JoVe: this site has a bunch of experimental protocols and video demonstrations – for basic experiments as well as super technical ones. https://www.jove.com/
AAT Bioquest Serial Dilution Calculator and Planner: this helps you plan out serial dilutions. It’s really helpful when you’re trying to figure out how much of what concentration you need to start when you’re using awkward dilution factors. You give it your starting concentration & dilution factor (e.g. 2 for a 1:2 dilution where you’re halving the concentration each time) OR you give it a concentration range. Then you give it your stock concentration, desired final volume for each dilution, & # of dilutions and it works out the math for you. https://www.aatbio.com/tools/serial-dilution
And here’s my post on serial dilutions: http://bit.ly/serialdilutions
The next couple of tools will basically do all the dimensional-analysis-ing for you. Make sure you know how to do it for yourself! http://bit.ly/dimensionalanalysising
Promega Biomath Calculators: they have calculators that will let you do various concentration conversions for DNA and proteins https://www.promega.com/resources/tools/biomath/?calc=dnaprotein
GraphPad Molarity Calculator: they have calculators for: mass from volume & concentration; volume from mass & concentration; molarity from mass & volume; diluting stock solutions https://www.graphpad.com/quickcalcs/Molarityform/
Sigma Calculators & Apps: there’s a TON of them… https://www.sigmaaldrich.com/US/en/support/calculators-and-apps
Good Calculators Significant Figures Calculator: it will do the calculations for you, but it also explains how to do them and offers a quiz https://goodcalculators.com/sig-fig-significant-figures-calculator/
more on what “sig figs” are here: http://bit.ly/significantfiguress
Wolfram Alpha: I haven’t really used it much since undergrad, but it was super helpful especially in math and physics because they work out solutions so you learn instead of just being fed the answers (but they give you those too 🙂 ). https://www.wolframalpha.com/
Chemspider: gives you more information than you ever wanted to know about any chemical! http://www.chemspider.com/
For people working with proteins… Protein analysis & prediction tools:
UniProt (this is your basic starting point for finding & learning about a protein – it connects you to way more resources through like a bazillion links & lets you align sequences): https://www.uniprot.org/
In that post I also talk about
ExPasy ProtParam (this lets you calculate pI, molecular weight, extinction coefficient, & more): https://web.expasy.org/protparam/
SOOOOOO useful when doing protein purifications! The pI can help you choose whether to use anion or cation exchange and what to use for the pH of your buffers. The molecular weight helps you know where to expect to see a band on your gel and what column to use for size exclusion (and what elution volume to expect).
PhosphoSitePlus: lets you find information about the post-translational modifications of a protein of interest. As the name suggests, it tells you about phosphorylation, but also things like acetylation, ubiquitinylation, & other modifications. Info includes strength of the evidence, putative suspects (modifying enzymes) etc. https://www.phosphosite.org/homeAction.action
PredictProtein: this free server will predict a lot of things about proteins based on their sequence. per their website, “PredictProtein integrates feature prediction for secondary structure, solvent accessibility, transmembrane helices, globular regions, coiled-coil regions, structural switch regions, B-values, disorder regions, intra-residue contacts, protein-protein and protein-DNA binding sites, sub-cellular localization, domain boundaries, beta-barrels, cysteine bonds, metal binding sites and disulphide bridges.” https://predictprotein.org/
Online Analysis Tools by Dr. Andrew M. Kropinski, Departments of Food Science; and, Pathobiology University of Guelph, Guelph, Ontario.
Links galore!. I especially like this page with links to a bunch of protein secondary structure tools (from predicting to drawing): https://molbiol-tools.ca/Protein_secondary_structure.htm Great for when you know there’s a software out there somewhere that will do what you want but you have no clue where!
Biologics International Corp Codon Usage Table: I use this when planning site-directed mutagenesis to introduce nucleotide changes to make amino acid changes in a protein I will recombinantly-express. Since there are often multiple codons to choose from for a specific amino acid, I try to use the one that’s preferred by the cells that I’m working with, unless that means making a greater # of nucleotide changes:
and here’s my post on site-directed mutagenesis: https://bit.ly/mutagenesisconstructs
and on codon usage bias & codon optimization: http://bit.ly/codonbias
Working with DNA & RNA
The Sequence Manipulation Suite: this site has a bunch of tools, but what I’ve used it most for is generating the reverse-complement, the reverse, and the complement of DNA sequences: https://www.bioinformatics.org/sms/index.html
Expasy Translate: this will translate a nucleic acid sequence to amino acids in the various reading frames: https://web.expasy.org/translate/
Watcut: a helpful tool for finding restriction enzyme sites in a DNA sequenceand opportunities to introduce silent mutations to introduce cut sites http://watcut.uwaterloo.ca/template.php⠀
Addgene repository: they serve as a repository where labs can deposit plasmids for distribution. So basically you can search add gene for a cDNA of interest and find it – often cloned into multiple backbone vectors you can choose from based on the desired expression system (though you can always subclone it out into one you want). They have over 104,000 and counting to choose from. www.addgene.org/browse/
If you care more about the vector, you can also search by that and get various expression vectors https://www.addgene.org/vector-database/
NCBI BLAST: the US National Institute of Health (NIH) has a National Center for Biotechnology Information. And they host a website called Basic Local Alignment Search Tool which compares biological sequences you put in to all (or a selected subset) of the sequences in the database. https://blast.ncbi.nlm.nih.gov/Blast.cgi
You can think of it a bit like 23andMe/Ancestry.com for biological molecules. There are a number of different versions of it depending on what you want to compare – nucleotides (DNA or RNA), protein sequences, etc. – and you can choose how stringent you want the comparison to be (e.g. do you only want to find perfect matches, or are you looking for some more evolutionarily-distant relatives?) The NCBI has a BLAST Program Selection Guide you can look at if you want all the details https://www.ncbi.nlm.nih.gov/blast/BLAST_guide.pdf But here’s the gist and when they may be helpful
nucleotide blast (aka blastn): this is for searching for/comparing nucleotide (DNA or RNA) sequences. I use this sometimes when I get back sequencing results from some of my cloned plasmids and I have absolutely no clue what was amplified… I can search for the amplicon and figure out where that sequence came from (often from somewhere else on the plasmid or from some contaminating bacterial genomic DNA)
blastx: this is for if you want to put in “translated nucleotides” and figure out what protein they correspond to – remember “x” for “exons” or “expressed.” With blastn, you are often searching genomic DNA – so parts that have protein-making instructions (coding parts aka exons) and parts that “just” have regulatory instructions (non-coding parts – introns (parts between exons that get removed during mRNA processing) and intergenic regions (parts between genes)). But with blastx, you’re putting in just protein-coding parts. For example, the sequence might correspond to a messenger RNA (mRNA), which is the edited RNA copy of a gene that is used by ribosomes to make the encoded protein in a process called translation. What blastx does is it takes the sequence you enter and “pretends it’s a ribosome” – it (in make-believe-land) translates all 6 possible open reading frames (ORFs) and then compares the resultant amino acid sequences to the amino acid sequences of known proteins.
Why might it be useful? Many amino acids can be spelled by multiple codons. So the codons can change while the protein itself stays the same and thus two DNA sequences might not look very similar even though the proteins look very similar. If you just used blastn you wouldn’t pick up on the similarity but you would find it with blastx. Conversely, if a sequence looks kinda similar at the DNA level but is in an intron or an intergenic region or is in a different reading frame in its natural context or something you could get “false hits” if you used blastx to try to find proteins.
It’s also useful if you want to detect coding regions in a sequence (genes aren’t obvious!). So say you have a long DNA sequence and you want to see what part of it actually has protein-making instructions. You could stick it in blastx and the protein-instruction parts will give you hits.
tblastn: this is the opposite of blastx. Here you’re taking a protein sequence and trying to find genes that code for it (or similar proteins). Maybe, for instance, you want to find the genes for versions of that protein in other species (homologs). So, if you put in EGADS, what it would find open reading frames that would make it. Problem is, since there are multiple spellings for each letter, there are lots of possible open reading frames, so what it does is, instead of trying to generate all the “what ifs” it generates the “whats” – it searches through a database of translated nucleic acids.
tblastx: search translated nucleotides for translated nucleotides
protein blast (blastp): this searches protein databases for protein sequences
miRBase: the microRNA database: the place to go if you want to search for a microRNA (miRNA). It gives you info about the sequence as well as how confident you can be that it’s legit and not a false positive (misannotation) (look to # of reads for a good indication). Note that human miRNAs will start with “hsa-“ and then miR-… https://www.mirbase.org/
Targetsscan: a database of predicted target sites for miRNAs along with info about confidence, whether there’s published experimental evidence, evolutionary conservation, etc. You can search a specific miRNA or miRNA family to find target genes or search a target gene to find miRNAs that target it. http://www.targetscan.org/vert_71/
RNAfold WebServer: predicts secondary structures of single stranded RNA or DNA sequences http://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuite/RNAfold.cgi
unpaywall: this browser extension will put a green lock icon on articles that have a (legal) freely available version somewhere on the web and direct you to it. https://unpaywall.org/products/extension
note: you can sometimes find (non-legal) versions with Sci-hub…
PubPeer: this can help you weed out really bad papers. PubPeer is a site where people can comment on papers. The comments can be good or bad, but most of the time these comments are drawing concerns about figures potentially containing altered images, etc. You can install a browser extension that will give a popup if articles on a page you’re on have comments on PubPeer. It will also tell you if articles have been amended or retracted. https://pubpeer.com/static/extensions
scite_: I use this browser extension that generates a sidebar which tells you the number of positive, neutral, and negative/questioning citations. When you click on it you get a report that shows you snippets of where it’s cited and links to see those full articles. In the free version you can’t filter the results or anything though. Especially as a trainee I think it’s really helpful in giving me a better idea of the wider context of any one work and how others in the field interpret the results. https://scite.ai/
make sure to use a reference management tool. There are lots of helpful ones like Mendeley (a free program I use), Zotero (also free), Endnote (not free), and Pages (not free). more on them here: https://bit.ly/referencesreference ; YouTube: https://youtu.be/VIds4W2nut8
Most of the time, I import citations using Mendeley’s web importer browser extension: https://www.mendeley.com/reference-management/web-importer
Adobe Illustrator: I use this vector graphics program for making all of my figures. More here: more on Adobe Illustrator & figure-making: https://bit.ly/geekygraphics & https://bit.ly/adobeillustratortips
a free alternative is InkScape: https://inkscape.org/
Illustrator for Biological Sequences (IBS): lets you make simple domain architecture graphics – you can enter domain start and end #s and it will illustrate them. helpful for making sure you get the scaling right! http://ibs.biocuckoo.org/
Introduction to structures:
The PDB based out of the US is the the Research Collaboratory of Structural Bioinformatics (RCSB) PDB has a great website called PDB-101: http://pdb101.rcsb.org/
If you go to this page in the “learn” tab, you’ll find this Introduction to PDB Data: http://pdb101.rcsb.org/learn/guide-to-understanding-pdb-data/introduction as well lots of pages about the basics of crystallography and crystal structures.
They also have lots of cool resources for teachers and learners, including downloadable infographics and posters and make-it-yourself paper models. I used the GFP model when I taught a summer camp lesson: https://bit.ly/gfpfunscience Structural biology can be kinda hard to get into, so they have ways to lure you in, like their “Molecule of the Month” series. Each month they feature a different molecule or class of molecules, tell you cool stuff about them, and walk you through some structures.
THE PDB!!!! This is where all the structures are deposited. https://www.rcsb.org/ more about the PDB and structures: blog: https://bit.ly/pdbstructures ; full video: https://youtu.be/NgXwP7gGPyA ; short video: https://youtu.be/1uKC08Z_lYQ
SBGrid is a consortium that labs can join that provides structural biology software. They also have weekly lectures that are free to watch on YouTube. They often have talks by software developers that can be very useful. https://www.youtube.com/channel/UCbo1TnKiXGtkE_R5b526JmQ
One of my favorites is on using ChimeraX’s augmented reality features!
SBGrid Consortium talk, “Making Augmented Reality Videos with ChimeraX”, by Tom Goddard, University of California, San Francisco, Resource for Biocomputing, Visualization, and Informatics https://youtu.be/g80hxLO1SVk
For visualizing and making pretty graphics of protein structures, there are 2 main tools, ChimeraX & PyMol. ChimeraX is fully free for academic use. PyMol has a free version with slightly limited functionality and academic labs often have a license for the full thing.
PyMol also has a really great wiki with tutorials. https://pymolwiki.org/index.php/Main_Page
A couple other software programs I used extensively in crystallography:
- Phenix: I used this for phasing, model building and automated refinement. http://www.phenix-online.org/ They also have some great tutorials on YouTube: https://www.youtube.com/c/phenixtutorials
- Coot: This is the software I used for manual refinement https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ This cheatsheet is a definite must! https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/web/docs/crib-sheet.pdf
- Those are integrated with each other, along with MolProbity which tells you about things like clashes and awkward geometry (and is developed by one of my heroes, Jane Richardson – more on her here: https://bit.ly/ribbonsandrichardson
3D bio notes: you put in the PDB code for a structure, then this site integrates a ton of information about the protein from various sources and lets you see sites of mutations, etc. in 3D. Also good for a quick overview or on the go look when you don’t want to open PyMol. http://3dbionotes.cnb.csic.es/ws
Crystallisation Construct Designer2: helps you design protein constructs to attempt to crystallize – good for figuring out where to make truncations & stuff (e.g. identify domain boundaries & remove predicted disordered regions at the end) https://ccd.rhpc.nki.nl/tutorial
MPI Bioinformatics Toolkit: this has lots of tools for structural prediction, homology finding, etc. https://toolkit.tuebingen.mpg.de/
SWISS-MODEL: I used this for homology modeling in undergrad. It’s good for when there’s a solved structure of a highly homologous protein to one you want to know the structure of (such as if there’s a structure of the mouse one but you’re interested in the human one) https://swissmodel.expasy.org/
Deep-learning protein structure prediction tools that try to predict any protein’s whole structure from the sequence. I did a post on them here: blog: https://bit.ly/proteinpredictionponderings ; YouTube: https://youtu.be/uKSgTdLxQQw
links for the David Baker lab’s Rosetta/robetta:
links for AlphaFold:
More specifically about crystallography:
Introduction to Macromolecular Crystallography, Second Edition, Alexander McPherson. First published:11 March 2008.Print ISBN:9780470185902 |Online ISBN:9780470391518 |DOI:10.1002/9780470391518
I had the pleasure of sitting in on the CSHL crystallography course twice and learning directly from Dr. McPherson (and a who’s who list of great crystallographers!). This book is really good and explains things in a comprehensive way that’s still comprehendible! It doesn’t get too into the math and instead helps you get a more intuitive sense of what’s going on. Highly recommend.
Crystallography Made Crystal Clear: A Guide for Users of Macromolecular Models, Gale Rhodes. ISBN 0080455549, 9780080455549 https://books.google.com/books/about/Crystallography_Made_Crystal_Clear.html?id=rwnR6qvaWgkC&source=kp_book_description
This is a more “meaty” book for those interested in really getting it down.
“Protein crystallography for non‐crystallographers, or how to get the best (but not more) from published macromolecular structures” by Alexander Wlodawer, Wladek Minor, Zbigniew Dauter, and Mariusz Jaskolski. https://febs.onlinelibrary.wiley.com/doi/full/10.1111/j.1742-4658.2007.06178.x
This is probably one of my most-reread articles. It does a great job explaining the basics of how to go about critically interpreting the quality of protein crystal structures, what to keep an eye out for, and what the basic statistics mean.
Resources for Readers of Crystallography Made Crystal Clear https://spdbv.vital-it.ch/TheMolecularLevel/CMCC/index.html
This goes along with that book, but you don’t need the book to appreciate it. It has links to TONS of useful software programs and articles for learning about crystallography
Protein Crystallography Course, Randy Read. Clear visuals & explanations. https://www-structmed.cimr.cam.ac.uk/course.html
If you want to get hard-core into the nitty gritty of protein crystallography, Dr. Andrea Thorn has a great YouTube series of videos, “Basics of Macromolecular Crystallography”: https://youtube.com/playlist?list=PLHHBmgJ8vFm6xZPlWlRGuBaoOM3OGlN5T
For cryo-EM, EM-University has hours and hours of content and resources. Basically a free full course (minus the hands-on stuff of course): https://em-learning.com/?CID=2019-LS-SPA-EMLearning&utm_source=Comms-Blog&utm_medium=EM+Blog&utm_campaign=2019-LS-SPA-EMLearning&utm_term=speak-to-an-expert-2
Some resources for people doing binding assays
The GraphPad Guide to Analyzing Radioligand Binding Data Dr. Harvey Motulsky http://www3.uah.es/farmamol/Public/GraphPad/radiolig.htm
Really helpful for analyzing experiments from slot blots!
more on slot blots: https://bit.ly/slotblotalot
Super helpful for understanding binding affinity concepts and making sure you set up your binding experiments in a way that will give you informative results.
How to measure and evaluate binding affinities. Inga Jarmoskaite, Ishraq AlSadhan, Pavanapuresan P Vaidyanathan, Daniel Herschlag. eLife 2020, DOI: 10.7554/eLife.57264 https://elifesciences.org/articles/57264#content
This is one of the most helpful articles basically ever…